Transgenic mice expressing mutant human APP and forming congo red staining plaques

ABSTRACT

Provided is a transgenic non-human eukaryotic animal whose germ cells and somatic cells contain the amyloid precursor protein sequence introduced into the animal, or an ancestor of the animal, at an embryonic stage. In mice, an age-related CNS disorder characterized by agitation, neophobia, seizures, inactivity, diminished cerebral glucose utilization, cortico-limbic gliosis, and death, develops. An acceleration of this disorder occurs in transgenic mice expressing human and mouse Alzheimer amyloid precursor proteins (APP) produced using a hamster prion protein gene-derived cosmid vector that confers position-independent, copy number-dependent expression. In transgenic mice the disorder develops in direct relationship to brain levels of transgenic APP, but mutant APP confers the phenotype at lower levels of expression than wild-type APP. The disorder occurs in the absence of extracellular amyloid deposition, indicating that some pathogenic activities of APP are dissociated from amyloid formation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a con of U.S. Ser. No. 08/664,872 filed Jun. 17,1996 now U.S. Pat. No. 5,877,399 is a continuation-in-part of U.S. Ser.No. 08/644,691 filed May 10, 1996, now abandoned which is acontinuation-in-part of U.S. Ser. No. 08/189,064 filed Jan. 27, 1994,now abandoned which disclosures are incorporated herein by reference.

NOTICE REGARDING FEDERAL FUNDING

This research was supported in parts by grants from the NationalInstitutes of Health, including grant number K08-NS01419. The governmentmay have rights in this invention.

INTRODUCTION

Technical Field

The invention relates to transgenic animals with progressive neurologicdisease characterized by both behavioral and neuropathological changesas compared to nontransgenic age-matched animals and their use forscreening for agents which can be used to treat or cure progressiveneurologic syndromes such as Alzheimer's disease. The invention isexemplified by transgenic mice which express native or mutant β-amyloidprecursor protein in brain tissue at superendogenous levels undercontrol of prion protein gene regulatory sequences.

Background

The term degenerative as applied to diseases of the nervous system isused to designate a group of disorders in which there is gradual,generally relentlessly progressing wasting away of structural elementsof the nervous system; many of the conditions so designated depend uponabnormal genetic factors. The degenerative diseases manifest themselvesby a number of syndromes distinguished by their clinical andpathological features. Nevertheless, there are certain aspects common toall. These aspects include a gradually progressive course of diseaseonset, bilaterally symmetric distribution of the changes brought aboutby the disease, and in many cases, the almost selective involvement ofanatomically or physiologically related systems of neurons. Typicallythe pathologic process is one of slow involution of nerve cell bodies ortheir prolongations as nerve-fibers.

Among the degenerative diseases of the nervous system are syndromes inwhich the outstanding feature is progressive dementia; the syndromes inthis group include senile dementia and Alzheimer's disease. Seniledementia is a fairly frequent condition of old age, not only in humansbut also in other animals. Alzheimer's disease is a pathologicallyidentical, but much more infrequent, progressive dementia which come sonwell before the senile period. The distinction between the twoconditions is purely clinical; pathologically they differ only in thatthe characteristic abnormalities tend to be more severe and widespreadin cases of Alzheimer's disease and to begin at an earlier age than atthe senile period.

Alzheimer's disease (AD) shows a slowly progressive mental deteriorationwith failure of memory, disorientation and confusion leading to profounddementia. The disease predominantly involves limbic and cortical regionsof the brain. There are several histologic features, but two arestriking. First, argyrophilic plaques containing the amyloidogenic Aβfragment of amyloid precursor protein (APP) are scattered throughout thecerebral cortex and hippocampus. Second, neurofibrillary tangles arefound in pyramidal neurons predominantly located in the neocortex,hippocampus, and nucleus basalis of Meynert. There are other changes,also. Granulovacuolar degeneration in the pyramidal cells of thehippocampus, which have been considered by some to be more specific forAD than plaques or neurofibrillary tangles, are observed. Finally, thereis neuronal loss and gliosis in the cortex and hippocampus.

There are patients with dementia who lack the pathologic features of AD(and therefore by definition have a different disease), and conversely,there are individuals with many of the pathologic features of AD whowere not demented prior to death. A diagnosis of AD requires that boththe clinical and the pathological features characteristic for thedisease be present in the patient; the diagnosis cannot be made withcertainty from either clinical or pathological features alone. Whetherneural dynsfunction and clinical abnormalities precede the developmentof the pathologic features, particularly the amyloid plaques andneurofibrillary tangles, is unknown.

The clinical manifestations of AD predict the regions of affected brainstructures in the forebrain, including the cerebral cortex, hippocampus,amygdala, and parahippocampal gyri. These regions are known as thecortico-limbic areas of the brain. The hindbrain is spared, includingthe cerebellum, the pontine and the medullary nuclei. Within thecerebral neocortex, the primary cortical area is relatively spared,which corresponds to the relative sparing of basic motor and sensorycortical functions observed clinically.

Research into progressive neurologic disorders such as AD, and means forscreening for agents which can be used to treat or cure these disorders,has been seriously impeded by the lack of easily accessible animalmodels. Some aspects of the neuropathology of aged primates are similarto those of human AD (Price, et al., (1992) J. Neurobiol, 23:1277-1294).Aged primates develop amyloid plaques and forme fruste neurofibrillarytangles. No other animals studied develop a disease resembling AD asclosely as do aged primates; aged primates are impractical to study inlarge numbers and their use raises both moral and economic issues.

Transgenic mice harboring APP transgenes have been described; however,the reported transgene product expression falls considerably short ofendogenous levels of APP; total APP levels in these order transgenicmice have not exceeded 150% of endogenous levels, and fails to generatea disease phenotype with a progressive neurobehavioral disorderaccompanied by pathology in the cortico-limbic regions of the brain. Inthese other transgenic mice, there have been no signs of progressiveneurologic disorder or of neuropathologic changes in the brain which maybe regarded as evidence of a true neurologic disease nor have changessuch as neurobehavioral changes which can be used in live animals as ameans of screening for agents which prevent, ameliorate or cure aprogressive neurologic disorder been described.

Missense point mutations in the gene coding for amyloid precursorproteins have been linked to familial AD. However, despite the discoveryof disease associated mutations in APP, most published attempts tocreate transgenic animals with AD have involved only wild-type APPtransgenes in mice (Kawabata, et al., (1991) Nature 354, 476-478; Quon,et al., (1991) Nature 352, 239-41; Wirak, et al., (1991) Science 253,323-325; Kammesheidt, et al., (1992) Proc Natl Acad Sci U.S.A. 89,10857-61; Lamb, et al., (1993) Nature Genetics 5, 22-30.) Unfortunately,several of the published studies purporting pathology have beenconfounded by inadequate documentation of transgene product expressionand/or misinterpretation of pathology. Two have been retracted(Kawabata, et al., (1991) Nature 354, 476-478; and Wirak, et al., (1991)Science 253, 323-325.

Previous efforts to create a model of AD in transgenic mice have beendiscouraging. In most cases, transgene product expression comparable toor exceeding endogenous levels of APP was not achieved and thetransgenes did not encode mutated APP. PCT/US92/11276 reports methodsfor using mutant genes. In some cases, the entire APP gene was notexpressed, just the carboxyl terminus (Kammesheidt, et al., (1992) ProcNatl Acad Sci U.S.A. 89, 10857-61); expression of only the carboxylterminus of APP may overlook any biologic effect that the rest of theAPP molecule may exert in AD.

Preamyloid APP plaques have been observed in some transgenic mice.However, preamyloid APP plaques are not necessarily indicative of adisease, since they are routinely observed in human brain regions, suchas the cerebellum, which are devoid of other signs of pathology orclinical manifestations. Increased APP immunoreactivity located withinvesicular structures in hippocampal neurons of transgenic mice has beenreported, but the significance of this immunoreactivity is unclear sincethe mice exhibited neither a progressive neurobehavioral disorder norevidence of true neuropathology.

In general, the ceaselessly progressive course of neurodegenerativediseases is uninfluenced by current treatment modalities. It thereforeis of interest to develop a transgenic non-human animal model fordegenerative neurologic diseases such as senile dementia and AD whereinthe animal develops a progressive degenerative neurologic disease of thecortico-limbic brain resembling the disease, both clinically andpathologically (e.g. the gliosis and the specific brain regionsaffected). It also is desirable that the animal develops neurologicdisease within a fairly short period of time from birth, facilitatingthe analysis of multigenerational pedigrees. The model can be used tostudy the pathogenesis and treatment of degenerative neurologic diseasessince there is a distinct and robust clinical and pathologic phenotypeto examine and score in the live animal.

RELEVANT LITERATURE

Transgenic mice (Swiss Webster x C57B6/DBA2 F1) expressing threeisoforms of mutant βAPPV717F with an overrepresentation ofKPI-containing isoforms show Alzheimer-type neuropathology includingabundant thioflavin S-positive Aβdeposits, neuritic plaques, synapticloss, astrocytosis and microgliosis (Games, et al., Nature 373:523-527(1995)), but deficits in memory and learning have not yet been reported.Transgenic mice (JU) expressing human wild-type βAPP751 show deficits inspatial reference and alternation tasks by 12 months of age (Moran, etal., Proc. Natl. Acad. Sci. USA 92:5341-5345 (1995)) but only 4% of aged(>>12 months) transgenic mice exhibited rare diffuse Aβ deposits that donot stain with Congo red dye (Higgins, et al., Annals of Neurology35:598-607 (1994)). Quon, et al. (1991) Nature 352:239 describetransgenic mice containing human amyloid precursor protein genes. Lamb,et al. (1993) Nature Genetics 5:22 describe transgenic mice in which theamount of amyloid precursor protein expressed is approximately 50% overendogenous levels. PCT application US92/11276 discloses methods forconstructing transgenic mice and rats which would express, under variouspromoters, three forms of the β-amyloid precursor protein (APP), APP695,APP751, and APP770. No data are provided in the specification as towhether APP expression is obtained in vivo using these methods. Also seeU.S. Pat. No. 5,455,169 and WO 9213069.

Other transgenic mouse studies of Alzheimer amyloid precursor (APP)protein expression include the following. Greenberg, (1993) Abstract421.12, Society for Neuroscience Abstracts 19:1035 discloses APP proteingene expression using MAPP and mMt-I promoters. Schwartz, et al. ((1993)Abstract 421.13, Society for Neuroscience Abstracts, 19:1035) discloseneuron-specific expression of human β-amyloid precursor protein (APP) intransgenic mice. Savage, et al. ((1993) Abstract 421.14 Society ofNeuroscience Abstracts 19:1035) disclose human amyloid precursor proteinexpression in transgenic mice as a model of Alzheimer's disease.Lieberburg, ((1993) Abstract 421.15, Science for Neuroscience Abstracts19:1035) disclose expression of human amyloid precursor protein intransgenic mice using the NSE promoter. Fukuchi, et al. ((1993) Abstract421.16, Society for Neuroscience Abstracts 19:1035) disclose intestinalβ-amylolidosis in transgenic mice. A chicken β-actin promoter and CMVenhancer were used for expressing the APP protein gene.

Wagner, et al. ((1983) Proc. Natl. Acad. Sci. U.S.A. 78:5016) describetransgenic mice containing human globin genes. Scott, et al. ((1989)Cell 59:847) describe transgenic mice containing hamster prion proteingenes. Hsiao, et al. ((1990) Science 250:1587) describe transgenic micecontaining mutant human prion protein genes. Hsiao disclosed a model forGerstmann-Straussler-Scheinker disease (GSS), a rare neurodegenerativedisease caused by mutations in the prion protein (PrP) gene, intransgenic mice in which levels of mutant transgene product exceedingendogenous levels were needed to generate a clinical and pathologicalphenotype (Hsiao, et al. (1990) Science 250:1587-1590); Hsiao, et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:9126-9130).

SUMMARY OF THE INVENTION

A transgenic non-human animal model for progressive neurologic diseaseis provided, together with methods and compositions for preparation ofthe animal model and methods for using it. The non-human mammals areobtained by the steps of introducing multiple copies of an expressioncassette into the non-human mammal at an embryonic stage, and developingthe embryo to term in a pseudo-pregnant foster female. The expressioncassette comprises an amyloid precursor protein coding sequence operablyjoined to regulatory sequences for expression of the coding sequence inneurologic tissues at a level at least two to four-fold that ofendogenous levels of wild-type amyloid precursor protein. The resultingtransgenic non-human mammals develop progressive neurologic disease inthe cortico-limbic areas of the brain. The transgenic animals find usefor example in screening protocols for agents which can be used fortreatment and/or prevention of progressive neurologic diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of a HuAPP cDNA sequence.

FIG. 2 is a diagrammatic representation of different APP sequences whichcan be expressed in transgenic animals (not exhaustive).

FIG. 3 is a diagrammatic representation of a hamster PrP cosmid vectorwith a tetracyclic-resistance sequence flanked by SalI sites replacingthe PrP coding sequence.

FIGS. 4 and 5 are diagrammatic representations of a hamster PrP cosmidvector fused with HuAPP sequences modified for strong translationinitiation as illustrated in FIGS. 6 and 7.

FIGS. 6 and 7 are diagrammatic representations of HuAPP sequencesmodified for strong translation initiation and flanking SalI restrictionsites.

FIG. 8 is a diagrammatic representation of PCR primers which can be usedto detect transgenes.

FIG. 9 shows age-related CNS dysfunction in transgenic andnon-transgenic FVB mice. In two lines of Transgenic mice,Tg(HuAPP695).TRImyc)1130H and Tg(HuAPP695.TRImyc) 1118 expressingvariant HuAPP at 3.6 and 1.4 times endogenous MoAPP levels,respectively, the average onset of illness was inversely related to APPlevels. A subset of Tg(HuAPP695.WTmyc) 1874 mice and non-Transgenic micedeveloped clinical and pathological abnormalities similar to those inaffected Transgenic mice, but with significantly lower penetrance at anygiven age.

FIG. 10 shows cortico-limbic hypertrophic astrocytic gliosis intransgenic and non-transgenic FVB mice exhibiting behavorialabnormalities. Coronal sections of cortico-limbic and brainstemstructures reacted with antibody to GFAP show hypertrophic gliosis incortico-limbic areas of animals exhibiting behavioral abnormalities.FIG. 10A, Tg(HuAPP695.TRImyc) 1118-334 exhibiting behavioralabnormalities (agitation and low corner index scores) at 144 days ofage, sacrificed at 206 days; FIG. 10B, non-Transgenic litter mate ofTg1118-334 without behavioral abnormalities, age 206 days; FIG. 10C,non-Transgenic #4565 exhibiting behavioral abnormalities (inactivity andlow corner index scores) at 324 days of age, sacrificed at 334 days;FIG. 10D, non-Transgenic litter mate of #4565 without behavioralabnormalities, age 334 days.

FIG. 11 shows transgenic HuAPP protein expression in brain tissue. HuAPPprotein expression was measured in a semi-quantitative fashion in fourlines of Transgenic mice, Tg(HuAPP695.WTmyc)466,Tg(HuAPP695.TRImyc)1056, Tg(HuAPP695.TRImyc) 1118,Tg(HuAPP695.TRImyc)1130H, harboring 40, 7, 21 and 74 transgene copynumbers, respectively. Relative levels of transgenic compared withendogenous brain MoAPP were examined by immunoblot analysis with twopolyclonal APP antisera, CT15 (FIG. 11A) and anti-GID (FIG. 11A), and amonoclonal antibody, 22C11 (FIG. 11B). CT15 antiserum recognized theC-terminal 15 amino acids of APP, a region in which mouse and human APPare homologous. GID antiserum recognizes an epitope 175-186 residuesfrom the amino terminus of APP695, a region in which mouse and human APPare identical. Equivalent amounts of protein from detergent-extractedbrain homogenates of non-Transgenic and Transgenic litter mates wereimmunoblotted in parallel. Primary antibody was revealed by ¹²⁵I-proteinA. For monoclonal antibodies, blots were first incubated with rabbitantiserum to mouse IgG. The amount of bound ¹²⁵I-protein A wasquantified using a phosphorimager, demonstrating a direct relationshipbetween transgene copy number and transgene product expression. Tomeasure the level of HuAPP specifically, brain homogenates were probedwith 6E10 antibody raised against residues 1-17 of human Aβ (Kim, et al.(1990) Neuroscience Research Communications, 7, 113-122). FIG. 11C showsthe regional expression of HuAPP in the brain. The relative amount ofHuAPP in 10% w/v homogenates of various tissues was specificallydetected in Tg(HuAPP695.TRImyc)1130H mice using 6E10 antibody.Equivalent amounts of protein were immunoblotted in each lane. Lanes 1,telencephalon; 2, diencephalon; 3, mesencephalon; 4, pons; 5,cerebellum; 6, medulla; 7, spinal cord. The highest HuAPP level, in thetelencephalon, was approximately twice that of the cerebellum.

FIG. 12 shows the dependence of transgenic brain APP expression uponspecies and copy number.

FIG. 13 shows HuAPP expression in neurons of transgenic mice. FIG. 13A.Tg, formic acid pretreatment, 6E10 antibody (hippocampus); FIG. 13B,Non-Transgenic, formic acid pretreatment, 6E10 antibody (hippocampus);FIG. 13C Tg, formic acid pretreatment, 6E10 antibody (cerebral cortex);FIG. 13D, AD plaque, formic acid pretreatment, 6E10 antibody; FIG. 13E,AD plaque, no formic acid pretreatment, 6E10 antibody; FIG. 13F, ADplaque, microwave pretreatment, 8E5 antibody; FIG. 13G, Tg, microwavepretreatment, 8E5 antibody (hippocampus); FIG. 13H, Non-transgenic,microwave pretreatment, 8E5 antibody (hippocampus).

FIG. 14 shows the dependence of the CNS disorder upon level oftransgenic brain APP expression and APP genotype.

FIG. 15A, the cosHaPrP.tet cosmid vector was used to drive expression ofhuman βAPP695 with the K670N-M671L mutation. The transgene used tocreate Tg2576 mice was made by substituting variant human βAPP ORF for atetracycline resistance cassette replacing the hamster PrP ORF locatedin the second exon. Exons are represented by thick black lines, 3′- and5′ untranslated regions by thick stippled lines. N=NotI, S=SalI. Methodsfor the creation of transgenes and transgenic mice, including Tg2576mice, are described in Hsiao, et al., (1995) Neuron 15: 1-16.

FIG. 15B, brain βAPP immunoblot of young and old transgene positive miceand non-transgenic control mice using 6E10 (21) which recognizes humanbut not mouse βAPP and 22C11 (Boehringer Mannheim) which recognizes bothhuman and mouse βAPP. Lanes 1-3: Non-transgenic mice; Lanes 4-6; 73day-old mice; lanes 7-8: 430 day-old mice. Detailed methods for βAPPquantitation are described in Hsiao, et al., (1995) Neuron 15: 1-16except that antibody binding was revealed using ³⁵S-protein A instead of¹²⁵I-protein A.

FIG. 16A, spatial alternation in a Y-maze. Transgene positive Tg2576mice exhibit significantly impaired spatial alternation at 10 months ofage but not three months of age. The methods used to perform this testare described in Hsiao, et al., (1995) Neuron 15: 1-16, except that theY-maze was opaque and animals were observed from an overhead camera toeliminate visual distraction posed by the tester. Stars indicatestatistical significance (t-test), p<0.05).

FIG. 16B, spatial reference learning and memory in the Morris water maze(Morris, (1984) J. Neurosci. Meth. 11:47) modified for use with mice.Transgene positive Tg2576 mice are able to learn and remember thelocation of the submerged platform at two and six months of age but showsignificant impairment by 9 to 10 months of age. Stars indicatestatistical significance (t-test, p<0.05).

FIG. 16C, spatial reference learning and memory in the Morris water mazein N2 Tg2576 mice retested at 12 to 15 months of age. Although transgenepositive mice were able to learn and remember the location of thesubmerged platform at two and six months of age, a subset of these miceshowed significant impairment when they were retested at 12 to 15 monthsof age. Thirty-six spatial training trials (9 trial blocks) and threeprobe trials were performed. The transgene positive mice showedsignificantly prolonged escape latencies after the 5^(th) trial blockand decreased platform crossings in both the second and third probetrials. Stars indicate statistical significance (t-test), p<0.05).

FIG. 16D, visually cued spatial reference test. Nine month-old transgenepositive Tg2576 mice performing poorly in the submerged platform mazeperformed as well as transgene negative animals in the visually cuedtest on the first trial day, indicating that their poor performance inthe submerged platform maze was due to neither visual nor motorimpairment. The consistently higher escape latencies on trial days 2through 4 may reflect more generalized cognitive impairment in thetransgenic mice. Stars indicate statistical significance (t-test,p<0.05).

FIG. 17, extracellular amyloid deposits in Tg2576 transgenic mice#A01493 (368 days) and #A01488 (354 days) overexpressing human βAPP695with the K670N-M671L mutation.

FIG. 17A, Tg2576-A01493, multiple plaques in the cerebral cortex andsubiculum staining with 4G8 monoclonal antibody, 10x magnification.

FIG. 17B, Tg2576-A01493, inset from panel A, 25x magnification.

FIG. 17C, Tg2576-A01488, plaque in subiculum staining with 4G8antibody,50x magnification.

FIG. 17D, Tg2576-A01488, plaque in section adjacent to panel C fails tostain with 4G8 antibody pre-absorbed with β(14-24).

FIG. 17E, Tg2576-A01488, plaques staining with thioflavin S.

FIG. 17F, Tg2576-A01488, plaque staining with β1 affinity purifiedantiserum specifically recognizing the amino-terminus of Aβ, 100xmagnification.

FIG. 17G, Tg2576-A01488, plaque staining with β42 affinity purifiedantiserum specifically recognizing the carboxyl terminus of Aβ(1-42),100x magnification.

FIG. 17H, Tg2576-A01488, plaque staining with β40 affinity purifiedantiserum specifically recognizing the carboxyl terminus of Aβ(1-40),50x magnification.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is directed to a transgenic non-human eukaryotic animal,preferably a rodent, such as a mouse, or other animal which is naturallyable to perform learning and memory tests, together with methods andcompositions for preparing and using the animal. The animal expresses anamyloid precursor protein (APP) sequence at a level in brain tissuessuch that the animal develops a progressive neurologic disorder within ashort period of time from birth, generally within a year from birth,preferably within 2 to 6 months, from birth. The APP protein sequence isintroduced into the animal, or an ancestor of the animal, at anembryonic stage, preferably the one cell, or fertilized oocyte, stage,and generally not later than about the 8-cell stage. The zygote orembryo is then developed to term in a pseudo-pregnant foster female. Theamyloid precursor protein genes are introduced into an animal embryo soas to be chromosomally incorporated in a state which results insuper-endogenous expression of the amyloid precursor protein and thedevelopment of a progressive neurologic disease in the cortico-limbicareas of the brain, areas of the brain which are prominently affected inprogressive neurologic disease states such as AD. The gliosis andclinical manifestations in affected transgenic animals are indicative ofa true neurologic disease. The progressive aspects of the neurologicdisease are characterized by diminished exploratory and/or locomotorbehavior and diminished 2-deoxyglucose uptake/utilization andhypertrophic gliosis in the cortico-limbic regions of the brain.Further, the changes that are seen are similar to those that are seen insome aging animals.

The present invention offers several advantages over existing models forprogressive neurologic disorders such as AD. The transgenic animalsexpress high levels of either native APP or mutant APP and develop aneurologic illness accompanied by premature death. Measurable changesare observed in these animals, including the neuropathological changessuch as gliosis and intracellular APP/Aβ accretions in the hippocampusand cerebral cortex and behavioral changes such as the diminishedexploratory behavior and impaired performance on learning and memorytests. The behavioral changes provide a particular advantage inscreening protocols for agents which can be used in a treatment forprogressive neurologic disorders such as Alzheimer's disease because theresults can be observed in live animals; it is unnecessary to wait untilthe animal is sacrificed to determine whether the agent is effective forits intended purpose.

Transgenic animals of the invention are constructed using an expressioncassette which includes in the 5′-3′ direction of transcription, atranscriptional and translational initiation region associated with geneexpression in brain tissue, DNA encoding a mutant or wild-type APPprotein, and a transcriptional and translational termination regionfunctional in the host animal. One or more introns also can be present.For expression, of particular interest are initiation regions (alsosometimes referred to as “promoters”) which provide for preferential orat least substantially specific expression in brain as compared to othertissue. By “at least substantially” is intended that expression in braintissue is greater than about 10 fold than in other tissue. Within thebrain, of particular interest is expression in the cortico-limbic area.The transcriptional initiation region can be endogenous to the hostanimal or foreign or exogenous to the host animal. By foreign isintended that the transcriptional initiation region is not found in thewild-type animal host into which the transcriptional initiation regionis introduced. By endogenous, is intended sequences both indigenous(i.e. natural to) the host animal and those present in the host animalas a result of an infectious disease, e.g. viral, prion, and the like.

A promoter from a gene expressed in brain tissue of the host animal isemployed for varying the phenotype of the host animal. Thetranscriptional level should be sufficient to provide an amount of RNAcapable of producing in a modified animal. By “modified animal” withinthe subject invention is meant an animal having a detectably differentphenotype from a non-transformed animal of the same species, forexample, one not having the transcriptional cassette including APPcoding sequences in its genome. Preferably, the promoter is a strongpromoter which drives a high level of expression of the APP codingsequence in brain tissue and/or which provides for many copies of thecoding sequence in brain tissue.

The promoter preferably comprises a transcriptional initiationregulatory region and translational initiation regulatory region ofuntranslated 5′ sequences, “ribosome binding sites”, responsible forbinding mRNA to ribosomes and translational initiation. Thetranscriptional initiation regulatory region may be composed ofcis-acting subdomains which activate or repress transcription inresponse to binding of transacting factors present in varying amounts indifferent cells. It is preferred that all of the transcription andtranslational functional elements of the initiation control region arederived from or obtainable from the same gene. In some embodiments, thepromoter is modified by the addition of sequences, such as enhancers, ordeletions of non-essential and/or undesired sequences. By “obtainable”is intended a promoter having a DNA sequence sufficiently similar tothat of a native promoter to provide for the desired specificity oftranscription of a DNA sequence of interest. It includes natural andsynthetic sequences as well as sequences which may be a combination ofsynthetic and natural sequences.

Tissue-specific transcription suggests that gene regulatory proteins arebound to enhancer sequences and other upstream promoter elements. Byenhancer element (“enhancer”) is intended a regulatory DNA sequence thatis capable of activating transcription from a promoter linked to it withsynthesis beginning at the normal RNA start site; which is capable ofoperating in both orientations (normal or flipped); and which functionseven when moved either upstream or downstream from the promoter. Bothenhancers and other upstream promoter elements bind sequences specificDNA binding proteins that mediate their effects. To identify the exactnucleotide sequences important for the function of the enhancer(s), andother upstream elements, fragments of the untranslated 5′-regionencoding a protein expressed in a tissue of interest are screened fortheir capacity to bind nuclear proteins and for their ability tofunction with a heterologous promoter. Binding experiments with nuclearproteins from brain tissue can be used to determine the presence ofenhancer and silencer sequences; the protein binding studies can be usedto pinpoint specific nucleotide sequences that bind to a correspondingseries of gene regulatory proteins.

The activity of each enhancer and other upstream promoter elementsgenerally is present on a segment of DNA which may contain binding sitesfor multiple proteins. The binding sites can generally be dissected bypreparing smaller mutated versions of the enhancer sequence joined to areporter gene whose product is easily measured. The effect of eachmutation on transcription can then be tested. Alternatively, fragmentsof this region can be prepared. Each of the mutated versions of theenhancer sequence or the fragments can be introduced into an appropriatehost cell and the efficiency of expression of a reporter gene measured.Those nucleotides required for enhancer function in this test are thenidentified as binding sites for specific proteins by means of gelmobility shift and DNA foot printing studies. An alternate means ofexamining the capability of isolated fragments of the region upstream ofthe promoter to enhance expression of the reporter gene is to look forsub-domains of the upstream region that are able to enhance expressionlevels from a test promoter which comprises the TATA CAAT box but showslittle or no detectable activity. A fragment of the 5′ region isinserted in front of the test promoter in an expression cassette, andthe effect on expression of the reporter gene evaluated. Of particularinterest for brain-specific, copy number-dependent expression areregions capable of binding to nuclear proteins in the region up to about20 kb from the mRNA start site of a brain-specific protein gene. Withinthis region, there may be several sub-domains of interest having thecharacteristics of brain specific enhancer elements which can beevaluated by using constructs.

A variety of promoter sequences an be used to control expression of APPcoding sequences. These include the metallothionine (MT) promoter fromwhich expression can be regulated through modulation of zinc andglucocorticoid hormone levels (Palmiter et al., Nature 300, 611-615(1982)); the rat neuron specific enolase gene promoter (Forss-Petter, etal., Neuron 5; 197—197 (1990)); the human β-actin gene promoter (Ray, etal., Genes and Development (1991) 5:2265-2273); the human plateletderived growth factor B (PDGF-B) chain gene promoter (Sasahara, et al.,Cell (1991) 64:217-227); the rat sodium channel gene promoter (Maue, etal., Neuron (1990) 4:223-231); the human copper-zinc superoxidedismutase gene promoter (Ceballos-Picot, et al., Brain Res. (1991)552:198-214); and promoters for members of the mammalian POU-domainregulatory gene family (Xi et al., (1989) Nature 340:35-42). ThePOU-domain is the region of similarly between the four mammaliantranscription factors Pit-1, Oct-1, Oct-2, and unc-86, and represents aportion of the DNA-binding domain. These promoters provide forexpression specifically within the neurons of transgenic animals.

Of particular interest as a transcriptional initiation region is onederived from a prion protein gene which is functional in the brain ofthe host animal. Prion protein is implicated in the pathogenesis andtransmission of Gerstmann-Straussler syndrome in humans and in scrapie,an equivalent non-human animal disease. Brain tissue serves as a sourcefor nucleic acid for preparing the desired sequences. To identify aprion promotor having the desired characteristics, where a prion proteinhas been or is isolated, it is partially sequenced, so that a probe canbe designated for identifying mRNA specific for prion protein. Sequenceswhich hybridize to the cDNA are isolated, manipulated, and the 5′untranslated region associated with the coding region isolated and usedin expression constructs to identify the transcriptional activity of the5′- untranslated region. As appropriate, sequences can be amplifiedusing PCR procedures known to those skilled in the art. In someinstances, a probe is employed directly for screening a genomic libraryand identifying sequences which hybridize to the probe. The sequenceswill be manipulated as described above to identify untranslated region.Prion promoter sequences are described in Basler, et al. (1986), Cell46:417-428 and Scott, et al. (1992) Protein Science 1:986-987.

The termination region which is employed primarily will be one ofconvenience, since the termination regions appear to be relativelyinterchangeable. The termination region may be native with thetranscriptional initiation region, may be native with the DNA sequenceof interest, or may be derived from another source. Convenienttermination regions are available from the prion protein gene.

The expression cassette which is used in the subject invention includespromoter and enhancer sequences from a gene which is expressed in thebrain and preferably which is expressed in a manner that is related tothe number of such sequences incorporated into the chromosome, namelythat higher transcription occurs with a larger number of transgenecopies incorporated into the chromosomes, operably joined to an APP genesequence and translational and transcriptional termination regions.Examples of promoter and enhancer sequences which are expressed in brainand which drive copy number dependent expression include the prionprotein promoter, such as that described by Scott, et al., ProteinScience (1992) 1:986-987, together with sequences upstream from thepromoter, because in order to obtain copy number dependent expression,it generally is necessary to include a sufficiently large region of DNAcontrolling transcription so that it is large enough to be relativelyunaffected by position effects. As an example, for the prion proteingene from hamster, approximately 20 kb of sequence upstream of thepromoter can be used.

As an example of construction of a cosmid vector for use in the instantinvention, components which are assembled, in the 5′ to 3′ direction ,include promoter and enhancer sequences of the prion protein gene, thecoding region of an APP gene sequence of interest and transcriptionaland translational termination sequences operably attached to a cosmidvector for delivery of the DNA constructs into the pronuclei of mouseeggs for expression of an APP gene in brain tissue. The enhancersequences may include a 20 kb region upstream of the prion proteinpromoter and may also include the noncoding exon 1 and the 10 kb introndownstream of exon 1 from the prion protein gene or can include thecoding sequence for more than one APP protein as described in, forexample, WO92/11276. Using molecular genetic techniques well known inthe art, the promoter/enhancer region of the prion protein gene may beisolated from a mammalian genomic cosmid clone used to create transgenicmice which express prion protein. The coding sequence of an APP gene isinserted between the promoter/enhancer region and the terminationsequences at unique restriction site or sites such that the codingsequence is translated in-frame. An APP protein in transgenic braintissue introduced using a cosmid vector as described above may beconfirmed to be at least two to four-fold that of endogenous levels. Amajor obstacle to the creation of a transgenic model of AD has been theinability to overexpress transgenic APP protein in the brain of thetransgenic animal. In some cases, mRNA is well expressed, but theprotein is poorly expressed. This indicates that the strength ofpromoters used may be adequate, but that protein translation may not beoptimal. Poor translation may result from a weak translation initiationsequence. Accordingly, it may be necessary to include a translationinitiation sequence wherein the positions at minus three and plus fourrelative to the initiation codon are A and G, respectively. See Table 1below.

TABLE 1 Transgene Translation Initiation Sequence OptimizationTranslation Transgene Initiation Sequence −3        +4Hacos.CS0HuAPP695-V717Imyc GCGATGCTG (SEQ ID NO:1) (native human APP)Hacos.CS1 ACCATGCTG (SEQ ID NO:2) Hacos.CS2 ACCATGGTG (SEQ ID NO:3)Hacos.MoAPP695-WT ACGATGCTG (SEQ ID NO:4) (native mouse APP)Hacos.MoPrP-P101L ATCATGGCG (SEQ ID NO:5) (native mouse PrP)

Any amyloid precursor protein sequence can be used to produce thetransgenic animals of the invention. An APP protein sequence, as theterm is used herein, means a sequence of the coding region of the APPgene which, when incorporated into the genome of the animal in multiplecopies and expressed in the transgenic animal at supraendogenous levels,promotes a progressive neurologic disease in the transgenic animal. Theneurologic disease is characterized by neurobehavioral disorder withgliosis and diminished glucose uptake and/or utilization incortico-limbic brain structures. The coding sequence can be from awild-type gene, or from a gene containing one or more mutations. Thecoding sequences can be a natural sequence or a synthetic sequence or acombination of natural and synthetic sequences. By mutant is intendedany APP which has an amino acid sequence which differs from that of thenative APP and includes substitutions, deletions, and the like. Bywild-type APP is intended native APP as it occurs in the relevant hostanimal.

Native human APP is encoded by a single 400-kb gene comprised of 18exons on chromosome 21. Alternative mRNA splicing gives rise to threeAPP isoforms. Two forms, APP751 and APP770 contain a Kunitz-proteaseinhibitor (KPI) region; the third, APP-695, lacks the KPI segment.Preferred sequences are those which are disease-linked. Examples ofdisease-linked mutations include a mutation at APP codon 693 (of APP770)linked to Dutch congophilic angiopathy (Levy, et al., (1990) Science248:1124), a mutation in APP linked to familial AD, valine-isoleucine atcodon 717 (of APP770) (Goate, et al., (1991) Nature 349:704-706), amutation wherein the valine at codon 717 is replaced by phenylalanine orglycine (Chartier-Harlin, et al., (1991) Nature 353: 844-846; Murrell,et al., (1991) Science 254: 97-99); and in one family with bothcongophilic angiopathy and AD, a mutation wherein alanine is replaced byglycine at codon 692 (Hendriks, et al., (1992) Nature Genetics1:218-221). In a Swedish kindred, a double mutation at codons 670 and671, resulting in a substitution of the normal lysine-methioninedipeptide by asparagine-leucine was found (Mullan, et al., (1992) NatureGenetics 1:345-347). APP with K670N-M671L is reported to be associatedwith increased Aβ1-40 secretion (Citron et al. (1992) Nature 360:672-674; Cai et al. (1993) Science 259: 514-516), while enhanced Aβ1-42production is reported for APP with the V717I mutation (Cai et al.(1993), supra; Suzuki et al. (1994) Science 264: 1335-1340). To obtainanimals with a progressive neurologic disease, while it can be used, itis unnecessary to use a coding sequence derived from an APP Gene with amutation at the 717 locus; likewise, while it can be used, it also isunnecessary to use a coding sequence which includes a KPI region and/orsplice sites within the coding region.

Table 2, below, lists some of the known amyloid precursor proteinsequences, some of which are genetically linked to familial Alzheimer'sdisease.

TABLE 2¹ Examples of APP Transgenes Translation APP ORF Size InitiationORF Species (Codons) Mutation CS1 or human, mouse 695 & V717I CS2 or 751or V717G human/mouse 770 V717F chimeras VVM717/721/722IAV KM670/671NL770A692G E693Q ¹The abbreviations used in Table 2 refer to the following:CS1 = translation initiation sequence as represented in FIG. 6; CS2 =translation initiation sequence as represented in FIG. 7; V = valine; I= isoleucine; G = glycine; F = phenylalanine; M = methionine; A =alanine; K = lysine; N = asparagine; L = leucine; E = glutamate; Q =glutamine; ORF = # open reading frame; numeral in the Mutation columnrefers to the mutated codon based upon the APP770 numbering system.

Of particular interest are novel chimeric APP genes, in which human Aβsequences replace the Aβ region of mouse APP. A158,5 is a 4-kDA peptidederived from APP. Examination of human (Hu), mouse (Mo), and chimeric(Mo/Hu) APP processing in mouse cell lines indicates that tangibledifferences are evident. HuAPP matures poorly in mouse cells, relativeto Mo- or combination Mo/HuAPP. However, the human Aβ sequences promotethe formation of soluble Aβ peptides that are normally produced.Mo/HuAPP chimeric protein matures more efficiently than HuAPP, andgenerates more soluble Aβ than MoAPP.

The animals used as a source of fertilized eggs cells or embryonic stemcells, the “host animal”, can be any animal, although generally thepreferred host animal is one which lends itself to multigenerationalstudies. Other preferred characteristics of the host animal include thatit is naturally able to perform learning and memory tests, and that itdoes not die at such an early age when it expresses high levels of APPthat there is insufficient time for observable behavioral and/orpathological changes to occur. Of particular interest are rodentsincluding mice, such as mice of the FVB strain and crossed commerciallyavailable strains such as the (C57B6) x (SJL.F1) hybrid and the (SwissWester) x (C57B16/DBA-z.F1) hybrid. The latter parental line also isreferred to as C57B16/D2. Other strains and cross-strains of animals canbe evaluated using the techniques described herein for suitability foruse as model for progressive neurologic diseases such as AD. In someinstances, however, a primate, for example, a rhesus monkey may bedesirable as the host animal, particularly for therapeutic testing.

Transgenic mammals are prepared in a number of ways. A transgenicorganism is one that has an extra or exogenous fragment of DNA in itsgenome. In order to achieve stable inheritance of the extra or exogenousDNA fragment, the integration event must occur in a cell type that cangive rise to functional germ cells, either sperm or oocytes. Two animalcell types that can form germ cells and into which DNA can be introducedreadily are fertilized egg cells and embryonic stem cells. Embryonicstem (ES) cells can be returned from in vitro culture to a “host”embryon where they become incorporated into the developing animal andcan give rise to transgenic cells in all tissues, including germ cells.The ES cells are transfected in culture and then the mutation istransmitted into the germline by injecting the cells into an embryo. Theanimals carrying mutated germ cells are then bred to produce transgenicoffspring.

A preferred method for making the subject transgenic animals is byzygote injection. This method is described, for example, in U.S. Pat.No. 4,736,866. The method involved injecting DNA into a fertilized egg,or zygote, and then allowing the egg to develop in a pseudo-pregnantmother. The zygote can be obtained using male and female animals of thesame strain or from male and female animals of different strains. Thetransgenic animal that is born is called a founder, and it is bred toproduce more animals with the same DNA insertion. In this method ofmaking transgenic animals, the new DNA typically randomly integratesinto the genome by a non-homologous recombination event. One to manythousands of copies of the DNA may integrate at one site in the genome.

Generally, the DNA is injected into one of the pronuclei, usually thelarger male pronucleus. The zygotes are then either transferred the sameday, or cultured overnight to form 2-cell embryos and then transferredinto the oviducts of pseudo-pregnant females. The animals born arescreened for the presence of the desired integrated DNA. By apseudo-pregnant female is intended a female in estrous who has matedwith a vasectomized male; she is competent to received embryos but doesnot contain any fertilized eggs. Pseudo-pregnant females are importantfor making transgenic animals since they serve as the surrogate motherfor embryos that have been injected with DNA or embryonic stem cells.

Putative founders are screened for presence of the transgene in severalways. Brain APP protein and APP expression are analyzed and thetransgene copy number and/or level of expression are determined usingmethods known to those of skill in the art. Brain APP protein RNAexpression, and transgene copy numbers are determined in weanlinganimals (4-5 weeks). When a promoter such as the prion protein genepromoter is used which is constituitively active in animals of weanlingage and older, it is not expected that there will be changes in levelsof transgenic APP RNA expression animals beyond weanling age. When adevelopmentally and/or tissue specific promoter is used, APP levels aremonitored to determine expression levels with age. The transgenicanimals also are observed for clinical changes. Examples ofneurobehavioral disorders for evaluation are poor mating response,agitation, diminished exploratory behavior in a novel setting,inactivity, seizures and premature death.

It is a theory of the invention that parameters that can influence thephenotype of transgenic animals include the host strain, the primarystructure of the APP and the levels of APP expression: the clinicalchanges observed in transgenic animals are a result of a combination ofthese factors. For a particular strain and a particular coding sequence,sufficient copies of an APP gene and/or a sufficient level of expressionof a coding sequence derived from a particular APP gene which willresult in observable clinical and/or behavioral symptoms, together witha measurable biochemical change in relevant brain structures can bedetermined empirically. By sufficient copies is intended that the totalexpression level from each construct is at least two-fold, preferably atleast two to four-fold, more preferably five-fold or greater than thatof an endogenous native gene, or that the overall copy number is such asto achieve this relative increase. In some instances, two to four copiesof the gene, especially of a mutated disease-linked gene, are sufficientto achieve a desired relative increase in APP, while in other instances,particularly where a native gene is used, a larger copy number may berequired. The copy number may range from five copies to more than 60copies, depending on the species of APP expressed and the particulardisease-associated mutations in the APP gene. As an example, theeffective range of copy numbers in FVB/N mice for HuAPP695.TRImyc isapproximately 20 to 75; for HuAPP695.SWE is approximately 30 to 50; andMoAPP.wt is greater than 25. In some instances a lower amount of APP iseffective in producing a progressive neurologic disorder, particularlywhere the mutation in the APP occurs in the Aβ region, or just upstreamof the Aβ region of the gene. Sufficient copies of a transgene thereforeis that number which produces expression of APP at a level which resultsin a progressive neurologic disorder.

The founder animals can be used to produce stable lines of transgenicanimals that superexpress APP, either mutant or native APP. For use ofpropagation, male founder mice are preferred. The animals are observedclinically. Analyses of transgene copy number (to exclude multipletransgene insertion sites), mRNA expression, protein expression,neuropathology, and glucose uptake in these animals are also performed.These studies provide information about the age of onset of illness, theduration of illness, the penetrance of the phenotype, the range ofneuropathologic findings, regional brain dysfunction, and the dependenceof phenotype upon levels of protein expression. Various changes inphenotype are of interest. These changes may include progressiveneurologic disease in the cortico-limbic areas of the brain expressedwithin a short period of the time from birth; increased levels ofexpression of an APP gene above endogenous expression levels and thedevelopment of a neurologic illness accompanied by premature death;gliosis and intracellular APP/Aβ accretions present in the hippocampusand cerebral cortex; progressive neurologic disease characterized bydiminished exploratory/locomoter behavior, impaired performance onmemory and learning tests, and diminished 2-deoxyglucoseuptake/utilization and hypertrophic gliosis in the cortico-limbicregions on the brain.

The animals also are screened using a species appropriateneurobehavioral test. For example, studies of locomotor/exploratorybehavior in mice is a standard means of assessing the neuropsychology(File and Wardill, (1975) Psychopharmacologia (Berl) 44:53-59; Loggi etal., (1991) Pharmacol. Biochem. Behav. 38:817-822). For example, formice the “corner index” (CI) is used. This is a quick and simpleneurobehavioral test to screen animals for evidence of brain pathology.The CI in transgenic mice which express mutant and wild-type APP is alsomeasured. A low CI (≦4) correlates with high mutant APP transgene copynumbers, premature death, and neuropathologic findings. The CI exhibitsa dosage dependent relationship to transgene copy number, which supportsthe validity of its use in accessing neurobehavioral sings in transgenicmice. The neuropathology of the animals also is evaluated. For rats, theMorris water maze test (described in Morris, (1984) J. Neurosci. Meth.11:47), is used. A modified version of this test can be used with mice.

Brain regions known to be affected by the syndrome of interest areparticularly reviewed for changes. When the disease of interest isAlzheimer's disease, the regions reviewed include the cortico-limbicregion, including APP/Aβ excretions, gliosis, changes in glucose uptakeand utilization and Aβ plaque formation. However, in strains of animalswhich are not long-lived, either naturally or when expressing highlevels of APP, not all behavioral and/or pathological changes associatedwith a particular disease may be observed. As an example, transgenicFVB/N mice expressing high levels of APP tend not to develop detectableAβ plaques, whereas longer lived C57B6/SJL F1 mice expressing identicaltransgenes do develop amyloid plaques which are readily detected withthioflavin S and Congo red. Immunologic studies of various brain regionsalso are used to detect transgene product.

The animals of the invention can be used as tester animals for materialsof interest, e.g. antioxidants such as Vitamin E or lazaroids, thoughtto confer protection against the development of AD. An animal is treatedwith the material of interest, and a reduced incidence or delayed onsetof neurologic disease, as compared to untreated animals, is detected asan indication of protection. The indices used preferably are those whichcan be detected in a live animal, such as changes in performance onlearning and memory tests. The effectiveness can be confirmed by effectson pathological changes when the animal dies or is sacrificed. Theanimals further can be used as tester animals for materials of interestthought to improve or cure Alzheimer's disease. An animal withneurologic disease is treated with the material of interest, and adelayed death, or improvement in neurobehavior, gliosis, or glucoseuptake/utilization, as compared to untreated animals with neurologicdisease, is detected as an indication of amelioration or cure.

The animals of the invention can be used to test a material orsituation, e.g. oxidants or head trauma, suspected of accelerating orprovoking Alzheimer's disease, by exposing the animal to the material orsituation and determining neurobehavioral decline, premature death,gliosis, and diminished glucose uptake/utilization as indicators of thecapacity of the test material or situation to induce Alzheimer'sdisease. The method further can include testing of therapeutic agents byexposing animals to a material or situation suspected of provokingAlzheimer's disease and evaluating the effect of the therapeutic agent.

Careful characterization of the transgenic animals should lead toelucidation of the pathogenesis of progressive neurologic syndromes suchas AD. The sequence of molecular events in mutant APP metabolism leadingto disease can be studied. The animals also are useful for studyingvarious proposed mechanisms of pathogenesis, including horizontaltransmission of disease (Prusiner, et al. (1987) Cell 63, 673-86),oxidation and free-radical production (Blass and Gibson, (1991) Rev.Neurol (Paris) 147:513-525; Ames et al., (1993) Proc. Nat'l. Acad. Sci.U.S.A. 90:7915-7922), inflammation (McGeer et al. (1993) Can. J. Neurol.Sci. 18:376-379, Rogers et al. (1992) Proc. Nat'l. Acad. Sci. U.S.A.89:10016-10020); neurotrophic factor deprivation (Perry, (1990)Alzheimer's Disease and Associated Disorders 4:1-13; Hefti andSchneider, (1991) Clinical Neuropharmacology 1:62-76); Koliatsoess etal., (1991) Ann. Neurol. 30:831-840), apolipoprotein E4 metabolism(Strittmatter et al., (1993) Proc. Nat'l. Acad. Sci. U.S.A.90:1977-1981), and potassium channel dysfunction (Etcheberrigaray, etal., (1993) Proc. Nat'l. Acad. Sci. U.S.A. 90:8209-8213). Such knowledgewould lead to better forms of treatment for neurologic disorders.

Other features and advantages of the invention will be apparent from thedescription of the preferred embodiments, and from the claims. Thefollowing examples are offered by way of illustration and not by way oflimitation.

EXAMPLES Example 1 PrP-HuAPP Transgene Construction

The human APP coding sequence was derived from a human cDNA (see Kang etal. (1987) Nature 325:733; Goldgabar et al., (1987); Science 235:877;Tanzi, et al., (1987) Science 235:880; and Robakis et al. (1987) Proc.Nat. Acad. Sci. U.S.A. 84:4190 and is illustrated in FIG. 1. It occursin three splice forms which are derived from a gene located onchromosome 21 as described by Kitaguchi et al. (1988) Nature 331:530;Tanzi et al. (1988) Nature 331:528; and Ponte et al. (1988) Nature331:525. FIG. 2 illustrates three features which may be incorporatedinto amyloid precursor protein sequences to produce the transgenicanimals of the invention: (1) splice form variants which result from thepresence or absence of the Kunitz protease inhibitor with or without theOX region; (2) amyloid precursor protein variants containing mutationswhich have been linked to illness in families with Alzheimer's diseaseas described by Goate (1991) Nature 349:704; Chartier-Harlin et al.(1991) Nature 353:844; Murell et al. (1991) Science 254:97; Hendriks etal. (1992) Nature Genetics 1:218; and Mullan et al. (1992) NatureGenetics 1:345, and families with congophilic angiopathy as described byLevy et al. (1990) Science 248:1124, and (3) a myc-tag at the carboxylterminus which can be used to facilitate immunodetection of transgeneproducts, but is preferably absent.

The required hamster prion protein gene functions were provided by ahamster prion protein cosmid vector in which a tetracycline-resistancesequence flanked by SalI sites replaces the prion protein codingsequence, as described by Scott et al. (1992) Protein Science 1:986. Thehamster prion protein cosmid vector is illustrated in FIG. 3. A 1.6 kbregion of DNA in the 3′-untranslated region of the prion protein gene isindicated as a useful probe for detecting transgenes made from thiscosmid.

The APP sequences and cosmid were used to construct the two fusion geneconstructions illustrated in FIGS. 4 and 5. The APP sequences weremodified for strong translation initiation, represented by theabbreviations CS1 and CS2. The constructions were made by substitutingthe SalI to KPNI DNA sequence at the 5′ end of the APP coding sequencefor DNA sequences made using the polymerase chain reaction (PCR) and twosets of primers. For the CS1 APP sequence illustrated in FIG. 6, theprimer set used was 5′-AAGTCGACACCATGCT GCCCGGTTTGGCACT-3′ (SEQ ID NO:6)and 5′-AAGGTACCTCCCAGCGCCCGAGCC-3′ (SEQ ID NO:6). For the CS2 APPsequence illustrated in FIG. 7, the primer set used was5′-AAAAAAGTCGACACCATGGTGCCCGGTTTGGCACT-3′ (SEQ ID NO:8) and5′-AAGGTACCTCCAGCGCCCGAGCC-3′ (SEQ ID NO:9).

Procedures were the conventional techniques described in Maniatis et al.(1982) Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory) and the polymerase chain reaction (PCR) described in Saikiet al. (1988) Science 239:487. The restriction sites shown in FIGS. 1-7are SalI (S), KpnI (K), BglII (B), XhoI (X) and NotI (N). The locationof the PCR oligomers used for detecting fusion constructs in animals areindicated by A and P in FIG. 8. Each PCR fragment synthesized for theconstructions was sequenced. The PCR fragments selected for use in theconstructions were free of unintended mutations.

The above PrP-APP cosmids were digested with NotI which releases thePrP-APP fusion gene from the pcos6EMBL vector illustrated in FIGS. 3-5.The Prp-APP fusion gene was isolated after size fractionation on anagarose gel and electroeluted. The PrP-APP fusion gene was furtherpurified in a series of organic extractions, includingphenol-chloroform, chloroform, and butanol, and precipitated in ammoniumacetate and ethanol. Prior to embryo injection, the PrP-APP fusion genewas dissolved in 10 mM Tris-Cl (pH 8.0) to a final concentration of 3-4μg/ml.

Example 2 Production of Transgenic Mice Containing PrP-HuAPP Transgene(APP Sequence VVM717/721/722IAV)

Each PrP-APP fusion gene was separately injected into fertilizedone-cell mouse eggs (Hogan et al. (1986) Manipulating the Mouse Embryo:A Laboratory Manual, Cold Spring Harbor Press, N.Y.; also see U.S. Pat.No. 4,736,866). Embryo donors and fertile studs were inbred FVB miceobtained from the National Cancer Institute (NCI); this resulted in theintegration of between 1 and 128 copies of PrP-APP fusion genes into thegenomes of the mice which developed to term. The injected eggs weretransferred to pseudo-pregnant foster females as described in Wagner etal. (1981) Proc. Nat'l. Acad. Sci. U.S.A. 78:5016. Mice were housed inan environmentally controlled facility maintained on a 10 hour dark: 14hour light cycle. The eggs in the foster females were allowed to developto term.

Example 3 Analysis of VVM717/721/722IAV Transgenic Mice

At four weeks of age, each pup born was analyzed in a PCR reaction usingDNA-taken from the tail. In each case, tail DNA was used as a templatefor a PCR reaction using the probes indicated at FIG. 8. The DNA foranalysis was extracted from the tail by the method described in Hanleyand Merlie (1991) Biotechniques 10:56. One μl of the tail DNApreparation (approximately 1 μg of DNA) was used to amplify a transgenespecific DNA fragment in a 25Z1 PCR reaction containing primers A and Pas illustrated in FIG. 8.

The PCR reactions indicated that 15 founder mice had retained aninjected PrP-APP fusion gene. The APP sequence in these animalscontained the VVM717/721/722IAV mutation and the myc-tag, but lacked theKPI/OX regions represented in FIG. 2. To determine transgene copynumber, denatured DNA in an exponentially diluted series was probed witha 1.6 kilobase (KB) radiolabelled segment of DNA from the3′-untranslated region of the hamster PrP gene as illustrated in FIG. 3.Among the founder mice with the highest transgene copy numbers(approximately 100 or more), two founder mice failed to breed, and athird founder sired offspring, which in turn failed to breed. Thus, the15 founder mice yielded 12 lines of transgenic offspring. A catalog oftransgenic founders with APP transgenes is shown in Table 3.

The founder animals were mated to uninjected animals and the DNA of theresulting 12 lines of transgenic offspring analyzed: this analysisindicated that in every case the injected genes were transmitted throughthe germline.

TABLE 3 Catalog of Transgenic Founders with APP Transgenes TransgeneProtein Animal ID Transgene Copy # Level Status Tg425LHacos.CS0HuAPP695-  1 Not Sac‘d V717Imyc detectable Tg466MHacos.CS0HuAPP695- 32-64 1.5-2X Alive WTmyc Tg1056L Hacos.CS1HuAPP695-16 Alive V717Imyc TG1057H Hacos.CS1HuAPP695-  64-128 Dead V717ImycTg1064L Hacos.CS1HuAPP695-  8 Alive V717Imyc Tg1072L Hacos.CS2HuAPP695- 1 Alive V717Imyc Tg1073L Hacos.CS2HuAPP695-  1 Alive V717Imyc Tg1118MHacos.CS1HuAPP695- 32-64 Alive V717Imyc Tg1119L Hacos.CS1HuAPP695-  1Alive V717Imyc Tg1123L Hacos.CS1HuAPP695-  1 Alive V717Imyc Tg1125LHacos.CS1HuAPP695-  8-16 Alive V717Imyc Tg1130H Hacos.CS1HuAPP695- 64-128 Sick V717Imyc Tg1135H Hacos.CS2HuAPP695-  64-128 Dead V717ImycTg1138H Hacos.CS2HuAPP695-  64-128 Dead V717Imyc Tg1140MHacos.CS2HuAPP695- 32-64 Alive V717Imyc

Six founder animals harbored >20 copies of the PrP-APP fusion genes. Allsix developed a neurologic disease characterized by progressivelydiminishing exploratory/locomotor behavior and premature death by fivemonths of age. In contrast, none of nine founder animals harboring <20copies of the PrP-APP fusion genes have developed the neurologic diseasewithin the first five months of age. The neurologic dysfunction wastransmitted to succeeding generations in an autosomal dominant fashion.

Expression of the newly acquired PrP-APP fusion genes in tissue wasdetermined by Western blot analysis using a monoclonal antibody, 6E10,raised to the first 17 residues of the human Aβ peptide (Kim, et al.(1990) Neuroscience Research Communicating 7:113-122). The fusion geneproduct was detected in the brain, spinal cord, skeletal muscle, heart,and, minimally, lung. It was not detected in the liver, spleen, kidney,or testis.

Expression of the PrP-APP fusion gene in brain tissue was quantitated byimmunodot blot analysis. Relative APP expression in brain tissue wascompared in transgenic and non-transgenic mice in an exponentiallydiluted series and reacted with antibody recognizing the 15 residues atthe carboxyl terminus of APP, CT15, which recognizes both mouse andhuman APP (Sisodia, et al (1993) J. Neurosciences 13:3136-3142). Thetotal APP protein in lines of mice which developed the neurologicdisease was at least 300% of endogenous levels. Where expression wasless than 300%, animals did not develop neurologic disease.

To obtain an index of brain function in affected transgenic mice,glucose utilization was regionally determined using a modification ofthe Sokoloff method described by Chmielowska et al. (1986) Exp. BrainRes. 63:607, which allows glucose uptake/metabolism in the mouse to bemeasured. Regional 2-deoxyglucose concentrations determineddensitometrically were normalized to the cerebellum, a region devoid ofpathology. Results in transgenic mice revealed significant reductions inglucose utilization of 20-30% in the hippocampus, amygdala, and someregions of the cerebral cortex as compared to age-matched non-transgeniclittermates.

Example 4 Analysis of Synthesis and Processing In Vitro

The synthesis and processing of the VVM717/721/722IAV mutant in culturedcells was examined to determine the effects of these mutations ondisease development. The wild-type HuAPP695myc and mutant cDNA geneswere cloned into the expression vector pEF-BOS (Osaka BioscienceInstitute, Osaka, Japan), then transiently transfected into mouseneuroblastoma cells, which were then continuously labeled with[³⁵S]methionine for 4 hours. Labeled APP molecules wereimmunoprecipitated with the monoclonal antibody 22C11 (Weidemann, et al.(1989) Cells 57:115-126). In extracts of cells, labeled APP molecules ofthe appropriate size were detected in similar levels. Media from thesecultures was examined for the presence of soluble APP fragments usingmAb 6E10 and mAb 4G8 (Kim, et al. (1990) supra.). Both of theseantibodies recognize the Aβ region of human APP. The mAb 6E10 recognizessequences in Aβ between Aβ1-17, while mAb 4G8 recognizes sequencesbetween Aβ1-28. The sequence of Aβ17-28 is identical to mouse Aβ andthus 4G8 cannot distinguish human and mouse APP. The media of culturestransfected with either gene contained a large ectodomain fragment ofAPP which is routinely observed.

One of the more recent discoveries relevant to the processing of APP hasbeen the detection of soluble Aβ 1-40 fragments in the medium ofcultured cells that express HuAPP. The Aβ fragments resemble peptidesfound in AD amyloid plaque lesions. Thus, it appears that APP isnormally processed into amyloidogenic fragments. Furthermore, mutationslinked to AD have been shown to alter the processing of APP to favor theproduction of soluble Aβ. To determine whether the VVM717/721/722IAVmutations affected the processing of APP, the culture medium wasexamined for small Aβ-containing APP peptides. An Aβ peptide fragmentthat was immunopurified by mAb 6E10 was prevalent in the media of cellstransfected with the mutant sequence. Similarly, the mAb 4G8 detectedincreased levels of Aβ peptide in the medium of cultures containing themutant.

An examination of cell extracts for accumulated APP fragments detectedincreased levels of a 20 kDa APP peptide fragment afterimmunoprecipitation with anti-myc polyclonal antiserum in cellsexpressing the mutant (FIG. 5C, line 3). Mutations generated in mutantHuAPP695myc affect the processing of the resultant APP product togenerate increased levels of soluble Aβ, and an intracellular C-terminalfragment of APP that is of sufficient length to include the Aβ region.Thus, the phenotype of animals created with the mutant APP is much likethat reported for humans expressing a mutant human APP gene that encodesmutations found in a Swedish kindred of AD. To date no investigatorshave reported increased production of Aβ as a result of expression ofHuAPP that encodes only the V642I AD-linked mutation (Golde et al.,(1993), Neuroscience Abstract 19:431, 182.7). However, this mutationappears to cause a change in the length of the soluble Aβ derivative,increasing it to Aβ1-42. Thus it appears that the VVM717/721/722IAVmutations are the primary cause of the increased production of solubleAβ. Studies of Aβ fibrillogenesis suggest that longer Aβ peptides aremore amyloidogenic.

Example 5 Comparison of Processing of Human and Mouse APP in Mouse Cells

Chimeric APP transgenes composed of mouse APP695 and human Aβ sequenceswere prepared and their processing evaluated. It is a hypothesis of theinvention that there are differences in the way mouse and human APP areprocessed in mice. To construct humanized MoAPP cDNA, a MoAPP gene wascloned and mutated to make it compatible with the cosSHaPrP.535 vector.Mouse cDNA was isolated by reverse transcriptase-polymerase chainreaction (RTPCR), and PCR primers included XhoI sites at the 5′ and 3′ends for cloning purposes. To remove an internal XhoI site in the mousecDNA, an additional primer was included that spanned the internal XhoIsite (codon 397) and contained a single base substitution thateliminated the XhoI site but preserved the correct amino acid sequence.The PCR product was subsequently sequenced to verify that unwantedmutations were not created in the PCR.

The Aβ region in HuAPP and MoAPP differs by three amino acid residues,which could affect the amyloidogenic potential of the transgene product.To humanize the mouse Aβ region, a segment of the HuAPP gene thatencompassed the Aβ region was amplified by PCR using primers thatinclude a sense primer that encompassed the BglII site at codon 590 ofHuAPP695 and an antisense primer that contained two point mutationscreating a NarI site at codon 626 (a cognate NarI site is found in theMoAPP cDNA), while maintaining the amino acid sequence (Table 4, primers1 and 2). This PCR product was digested with BglII and NarI and thencloned into the BglII and NarI sites of the MoAPP cDNA.

The chimeric (Mo/HuAPP) cDNA was sequenced across the BglII and NarIsites to verify that this region now contained human Aβ sequences and toverify that no other unwanted mutations were generated. To verify thatthis recombinant cDNA could be expressed into full-length protein, DNAwas cloned into a modified pEFBOS vector. The pEFBOS vector contains thepromoter element, first exon, first intron, and part of the second exonof the mammalian elongation factor 2 along with an SV40 origin ofreplication, permitting the replication of vectors and the highexpression of genes in COS-1 cells. COS-1 cells were transfected withpEF-BOSMo/HuAPP695 and cell extracts were analyzed by immunoblotting.CT15 recognized a full-length Mo/HuAPP polypeptide, whereasimmunostaining with monoclonal antibody 6E10 verified that the humanizedmouse cDNA product did indeed encode human Aβ sequences.

To generate chimeric Mo/HuAPP cDNA that encodes a double mutation linkedto an early-onset AD, a PCR-based approach similar to that outlinedabove using primers 2 and 3 (Table 4) was employed. The template for thereactions was a cloned copy of Mo/HuAPP695. The mutated chimeric genewas sequenced across the BglII and NarI sites to verify the presence ofmutations and to be certain that no unwanted mutations existed in thetransgene. The mutant Mo/HuAPP cDNA was cloned into pEFBOS andtransfected into COS-1 cells to determine whether APP polypeptides weresynthesized. An APP polypeptide of the predicted size reacted with bothCT15 and 6E10 antibodies.

An examination of the synthesis and processing of Mo-, Hu-, and Mo/HuAPPin mouse N2a cells has surprisingly revealed discernible differences.What is evident is that a greater percentage of MoAPP is cleaved togenerate a soluble ectodomain fragment than is HuAPP. The ratio ofcell-associated versus soluble MoAPP is approximately 1 to 5, while 3times more of the HuAPP is cell-associated than is soluble. Thepercentage of Mo/HuAPP695 that is cleaved to generate solubleectofragments appears to fall between that of Mo- and HuAPP as the ratioof cell-associated to soluble Mo/HuAPP approaches 1 to 1. The majorityof soluble APP ectofragments appear to arise from a cleavage eventwithin AD at the cell surface; the differences in the ratio ofcell-associated APP versus soluble ectofragments indicate differences inthe maturation of the polypeptides. Specifically, the majority of MoAPPreaches the cell surface and is cleaved by a secretase. In contrast,HuAPP may not reach the cell surface as efficiently, thus precludingsecretase cleavage. The Mo/HuAPP polypeptide appears to be intermediatebetween Mo and HuAPP. Alternatively, it is possible that sequenceswithin the Aβ domain influence the efficiency of secretase cleavage.

In addition to differences in the production of soluble APPectofragments, differences in the level of soluble Aβ peptides werenoted. All three proteins gave rise to soluble Aβ peptides that were ofa size and character consistent with identification as Aβ1-40. In cellstransfected with MoAPP, a fragment that is of a size and characterconsistent with identification as Aβ17-40 was detected. The Aβ17-40fragment is thought to arise after membranal cleavage of APP by theputative secretase, which cleaves between Aβ16 and −17. Only the Hu- andMoHuAPP derived Aβ1-40 peptides were recognized by mAb6E10 as expected.While MoAPP appeared to give rise to relatively equal amounts of Aβ1-40and Aβ17-40, HuAPP and Mo/HuAPP were preferentially cleaved to generateonly Aβ1-40. These results suggest that sequences differences within thehuman Aβ domain influence APP proteolytic cleavage.

TABLE 4 Primers Used In Constructing Recombinant APP Genes Primer SenseSequence Cloning Sites Codon Mutation 1 + CCGAGATCTCTGAAGTGAAGATGGATGBgl II none (SEQ ID NO:10) 2 − AAGCTTGGCGCCTTTGTTTGAACCCAC Nar I  none(SEQ ID NO:11) 3 + CCGAGATCTCTGAAGTGAATCTGGATGC Bgl II FAD (N595,L596)(SEQ ID NO:12)

Example 6 Comparison of Normal Aged Mice and Transgenic Mice

Transgene Construction

The PrP-APP transgenes were generated as described in Example 1 byreplacing a SalI-flanked tetracycline resistance sequence in a hamsterPrP cosmid vector (Scott et al., (1992), supra), with SalI-flanked humanand mouse APP coding sequences. Transgenic mice were prepared using oneof six different PrP/APP chimeric transgenes: murine wild-type APP695(MoAPP695.WT); human APP695 containing two mutations at K670N and M671L(APP770 numbering) (HuAPP695.SWE); human APP695 containing two mutationat M670N and K671L (APP770 numbering) (HuAPP695.SWE); human APP695containing a mutation at E693Q (HuAPP695.DUT); human APP770 with K670Nand M671L (HuAPP770.SWE); human APP695 with a triple mutation at V717I,V721IA, and M722V with a 3′-myc tag (HuAPP695.TRImyc); and humanwild-type APP695 with a 3′-myc tag (HuAPP695.WTmyc). TheSC1HuAPP695.SWE, CS1HuAPP770.SWE, CS1HuAPP695.TRImyc andCS2HuAPP695.TRImyc APP sequences were modified for strong translationinitiation.

Like the Swedish mutation, triple V7171I, V721A and M722V mutations inthe transmembrane domain of APP enhance secretion of Aβ by five-fold incultured cells. The 3′-myc tag, a 12 codon segment of the c-mycproto-oncogene, was shown in cultured cells to facilitateimmunodetection of transfection products (Wong and Cleveland, (1990) TheJournal of Cell Biology 111, 1987-2003). In Tg(HuAPP695.WTmyc) andTg(HuAPP695.TRImyc) mice the myc-tag was not as clearly detectable inWestern blots and histologic samples as HuAPP reacted withhuman-specific APP antibodies. The myc-tag exerted no apparent effect onthe phenotype, since Tg(HuAPP695.SWE, Tg(HuAPP770.SWE), andTg(HuAPP695.DUT) mice lacking the myc-tag develop the same clinical andpathologic abnormalities. The constructions were made by substitutingthe SalI to KpnI DNA sequence at the 5′ end of the APP coding sequencefor DNA sequences made using the polymerase chain reaction (PCR) and twosets of primers. For the CS1 APP sequence, the primer set used was5′-AAGTCGACACCATGCTGCCCGGTTTGGCACT-3′ and 5′AAGGTACCTCCCAGCGCCCGAGCC-3′.For the CS2 APP sequence, the primer set used was5′AAAAAAGTCGACACCATGGTGCCCGGTTTGGCACT-3′ (SEQ ID NO:8) and5′-AAGGTACCTCCAGCGCCCGAGCC-3′ (SEQ ID NO:7). The HuAPP mutations weremade using standard methods of site-directed mutagenesis. Each PCRfragment synthesized for the constructions was sequenced. The PCRfragments selected for use in the constructions were free of unintendedmutations. The PrP-APP cosmids were digested with NotI (which releasesthe PrP-APP fusion gene from the pcos6EMBL vector). The Prp-APP fusiongenes were isolated after size fractionation on an agarose gel andelectroeluted. The PrP-APP fusion gene was further purified with organicsolvents, and precipitated in ammonium acetate and ethanol. The PrP-APPfusion genes were dissolved in 10 mM Tris-Cl (pH 8.0) to a finalconcentration of 3-4 μg/ml prior to embryo injection. 1503:5′-CTGACCACTCGACCAGGTTCTGGGT-3′ (SEQ ID NO:13) and 1502:5′-GTGGATAACCCCTCCCCCAGCCTAGACCA-3′ (SEQ ID NO:14), located in the 3′region of APP and the 3′-untranslated region of PrP, respectively. The1503 primer recognizes a region which is homologous in mouse and humanAPP, and can therefore be used to detect both PrP-MoAPP and PrP-HuAPPDNA. Using primers 1502 and 1502: 5′-AAGCGGCCAAAGCCTGGAGGGTGGAACA-3′(SEQ ID NO:15), a parallel PCR reaction amplifying a fragment of murinePrP was performed as a positive control.

Transgene copy number analysis was performed using 5 μg denaturedpurified tail DNA baked onto nitrocellulose and hybridized to aradiolabelled 1.3 kb SalI-XhoI DNA fragment encoding a segment of thehamster PrP 3′-untranslated region located in the DNA sequence at the 5′end of the APP coding sequence for DNA sequences made using thepolymerase chain reaction (PCR) and the two sets of primers described inExample 1. The HuAPP mutation were made using standard methods ofsite-directed mutagenesis. Each PCR fragment synthesized for theconstructions was sequenced. The PCR fragments selected for use in theconstruction were free of unintended mutations. The PrP-APP cosmids weredigested with NotI and the Prp-APP fusion genes were isolated after sizefractionation on an agarose gel and electroeluted and further purifiesas described in Example 1. The PrP-APP fusion genes were dissolved in 10mM Tris-Cl (pH8.0) to a final concentration of 3-4 μg/ml prior to embryoinjection.

Transgenic Mouse Generation and Screening

Transgenic lines were initiated by microinjection of single-cell mouseembryos as described (Hogan et al., (1986) supra). Embryo donors andfertile studs were inbred FVB mice obtained from the National CancerInstitute (NCI). Post-weaning tail biopsy DNA was generated as described(Hanley and Merlie, (1991) Biotechniques 10, 56). One microliter of theunpurified DNA was used in a 25 μl PCR reaction. To detect PrP-APPfusion DNA, the PrP-APP fusion DNA was amplified using the polymerasechain reaction with a pair of oligomer primers, 1503:5′-CTGACCACTCGACCAGGTTCTGGGT-3′ (SEQ ID NO:13) and 1502:5′-GTGGATAACCCCTCCCCCAGCCTAGACCA-3′ (SEQ ID NO:14), located in the 3′region of APP and the 3′-untranslated region of PrP, respectively. The1503 primer recognizes a region which is homologous in mouse and humanAPP, and could therefore be used to detect both PrP-MoAPP and PrP-HuAPPDNA. Using primers 1502 and 1501: 5′AAGCGGCCAAAGCCTGGAGGGTGGAACA-3′ (SEQID NO:15), a parallel PCR reaction amplifying a fragment of murine PrPwas performed as a positive control.

Transgene copy number analysis was performed using 5 μg denaturedpurified tail DNA baked onto nitrocellulose and hybridized to aradiolabelled 1.3 kb SalI-XhoI DNA fragment encoding a segment of thehamster PrP 3′-untranslated region located in the hamster PrP cosmidvector (Scott, et al., (1992) supra). After two high-stringency washesand exposure to radiosensitive film, the relative intensities of signalsfrom genomic DNAs to transgenic mice and hamsters were compared using aphosphorimager to obtain transgene copy numbers relative to diploidhamster genomic DNA.

Analysis of Transgene Expression

APP transgene product expression was examined in progeny of transgenicfounders sacrificed at one to four months of age. Quantitativeimmunoblotting of extracts from brain homogenates was carried out inparallel with extract prepared from age-matched nontransgeniclittermates. 20% (w/v) homogenates of brain tissues were prepared in TNE(50 mM Tris-Cl pH 8.0, 150 mM NaCl, 5 mM EDTA with 2% PMSF) buffer,using a hand-held polytron. Homogenates were diluted with an equalvolume of TNE 1% N40, 1% deoxycholate, 0.4% SDS and sonicated in a bathsonicator until all viscosity was lost. Homogenates were then boiled for10 minutes and centrifuged at 10,000×g for 10 minutes.

The supernatants were mixed with an equal volume of 2 X sample buffer(Laemmli, (1970) Nature 227, 680-685), boiled 2 min., and fractionatedusing a 6% SDS-PAGE. Proteins were electrophoretically transferred toImmobilon membranes (Pierce) and incubated with polyclonal (CT15 andantiGID) and monoclonal (22C11 and 6E10) APP antibodies. Reactive rabbitpolyclonal antibodies were visualized following incubation withsecondary rabbit antibodies to mouse IgG before incubation with¹²⁵I-protein. Radiointensities were quantified on a phosphorimager(Molecular Dynamics, Inc.). APP expression in brain tissue was measuredin transgenic mice harboring different transgene copy numbers byquantification of immunoblots in transgenic lines with three antibodiesrecognizing both MoAPP and HuAPP, CT15 (FIG. 11), anti-GID (FIG. 11),and 22C11 (FIG. 11). CT15 (Sisodia et al., (1993) J. Neurosciences13:3136-3142; Borchelt et al., (1994) J. Biol. Chem 269: 14711-14714);anti-GID (Cole et al., (1989) Brain Res. Reviews 13:325-349); and 22C11(Weidemann et al., (1989) Cell 57:115-126) recognize both mouse andhuman APP equally, but 22C11 also binds APLP2, a close relative of APP,with the same avidity (Slunt et al., (1994) J. Biol. Chem269:2637-2644). Minor variations in HuAPP levels relative to MoAPPexpression obtained with different antibodies may reflect differences inthe avidity of antibody binding or distinctions in post-translationalprocessing between wild-type and variant HuAPP. Transgenic brain APPprotein expression was dependent upon copy number as well as the speciesof APP expressed (FIG. 12). Relative to HuAPP, equivalent levels ofMoAPP were achieved with lower numbers of transgene copies.

To measure the level of HuAPP specifically, brain homogenates wereprobed with 6E10 antibody raised against residues 1-17 of human Aβ (Kimet al., (1990) Neuroscience Res. Comm. 7:113-122). No reactivity to˜100-125 kD APP molecules was detected in non-transgenic mice (FIG. 11).In Tg1130H mice the highest levels of HuAPP detected on immunoblotsusing 6E10 antibody were in the brain and spinal cord, and much smalleramounts (<5% of brain levels) were found in the striated muscle, heart,skin, and lung. HuAPP was poorly detected or absent in the thymus,liver, spleen, kidney, testis, and small intestine.

Specific immunostaining for human APP/Aβ using the 6E10 or 8E5 antibody(Athena Neurosciences) revealed HuAPP throughout the brain. 8E5recognizes a segment of APP spanning residues 444-592 (APP695numbering). Two different methods were used to enhance APPimmunoreactivity in brain tissue from transgenic lines overexpressingHuAPP. In high copy number lines, following either formic acidpretreatment of tissue using 1:5000 dilution of 6E10 antibody ormicrowave pretreatment of tissue using either 1:100 6E10 antibody or1:100 8E5 antibody, APP staining was invariably present within vesicularstructures in large pyramidal cells of the hippocampus, parahippocampalarea, amygdala, and the cerebral cortex (FIGS. 13A, C, H). In somebrains, fainter immunoreactivity was also visible in smaller neurons inthe cortico-limbic regions of the brain and in large and small neuronsof the basal ganglia, brainstem, and cerebellum. Staining was absent innon-transgenic mice (FIGS. 13B, H) and in untreated brain tissue fromaffected transgenic mice. The pattern of HuAPP immunostaining obtainedreflected the widespread expression of HuAPP in the brain with thehighest levels of expression in the telencephalon, as independentlyconfirmed in regional brain immunoblots using the 6E10 antibody (FIG.11).

The 8E5 antibody stained amyloid plaques and intraneuronal vesicularstructures in microwaved tissue sections from patients with AD (FIG.13F). At 1:5000 dilution, the 6E10 antibody stained amyloid plaques frompatients with AD only after formic acid pretreatment of brain tissue(FIGS. 13D, E). However, in TgHuAPP mice neither the microwave norformic acid pretreatment of brain tissue revealed HuAPP stainingresembling extracellular amyloid or pre-amyloid deposits using eitherantibody. The abnormal phenotype in these transgenic mice, therefore,was not caused by amyloid or pre-amyloid deposition.

To assess the relative effects of mutant and wild-type APP transgeneexpression on the development of a CNS disorder, the percentage ofanimals sick or dead at 100 and 200 days in lines expressing differentlevels of wild-type HuAPP, mutant HuAPP, or wild-type MoAPP (Table 5)was determined. These data demonstrate a direct relationship between APPexpression and the development of an abnormal phenotype (FIG. 15). Acomparison of transgenic mice expressing wild-type HuAPP and mutantHuAPP was not possible over the full range of APP expression. However, acomparison of transgenic mice expression approximately two to four foldmutant HuAPP, (TgHuAPP695.TRImyc)1140 and (TgHuAPP695.TRImyc)1130, withtransgenic mice expressing approximately three fold wild-type MoAPP,(TgMoAPP695.WT)1874, indicates that mutant HuAPP will readily provokethe abnormal phenotype. This observation argues against the abnormalphenotype being due to a non-specific effect of transgenic protein overexpression, since mutant HuAPP conferred the disorder with higherpenetrance than wild-type MoAPP, demonstrating a specific effect of thetransgenic protein species it expressed. These data are represented astitration curves that demonstrate a direct relationship between APPexpression and the development of an abnormal phenotype (see FIG. 15).However, the left-shifted curve for transgenic mice expressing mutantAPP relative to wild-type APP indicates that expression of the mutantAPP more readily provokes the abnormal phenotype.

To ensure that overexpression of a foreign (human) species of proteindid not artefactually produce the abnormal phenotype, transgenic miceoverexpressing wild-type MoAPP were generated. In transgenic mice withMoAPP levels equivalent to 3.1-fold endogenous APP levels the samephenotype occurred, indicating that the observed phenotype was not dueto overexpression of a foreign species of protein.

TABLE 5 Clinical and pathological features of FVB mice expressing APPtransgenes Cortico- Copy limbic Extra- Number Transgenic % sick orgliosis cellular Weight (mean ± brain APP dead at 100 % sick or dead (inaffect- HuAPP (gm ± SEM) Line SEM) (mean ± SEM) days at 200 days edmice) deposits Tg Non-Tg Tg (HuAPP695. TRImyc) 1072 <0.05 0 (n = 21) 0(n = 21) Tg (HuAPP695. TRImyc) 1056  7 ± 1.7 0.3 ± 0.09 0 (n − 10) 10(1/10) Tg (HuAPP695. TRImyc) 1118 21 ± 3.7 1.4 ± 0.17 42.5 (20/47) 97(20/30) +(1/1) — 21.9 ± 0.46 Tg (HuAPP695. TRImyc) 1140 49 ± 2.5 2.0 ±0.32 83 (15/18) 93 (15/16) 18.0 ± 0.92 20.6 ± 0.38 Tg (HuAPP695. TRImyc)1130H 74 ± 3.7 3.6 ± 0.54 98 (59/60) 100 (60/50) +(3/3) — 11.4 ± 0.50 Tg(HuAPP695. TRImyc) 1057F  64 − 128 N/A 100 (1/1) 100 (1/1) +(1/1) — Tg(HuAPP695. TRImyc) 1138  64 − 128 N/A 100 (4/4) 100 (4/4) +(2/2) Tg(HuAPP695. SWE) 2123H 46 N/A 100 (2/2) 100 (2/2) Tg (HuAPP695. SWE)1844F 42 N/A 100 (1/1) 100 (1/1) +(1/1) — Tg (HuAPP695. SWE) 1837F 42N/A 100 (1/1) 100 (1/1) +(1/1) — Tg (HuAPP695. SWE) 1827F 59 N/A 100(1/1) 100 (1/1) +(1/1) — Tg (HuAPP695. SWE) 1665F 244  N/A 100 (1/1) 100(1/1) +(1/1) — Tg (HuAPP695. DUT) 2012 47 ± 3.4 N/A 100 (3/3) 100 (3/3)   ( /2) Tg (HuAPP695. WTmyc) 6214  4 ± 0.3 0 (n = 25) 0 (n = 25) Tg(HuAPP695. WTmyc) 466 40 ± 9.0 1.0 ± 0.21 0 (n = 12) 33 (1/3) −(0/1) Tg(HuAPP695. WTmyc) 6209 28 ± 6.1 1.6 ± 0.43 35 (5/15) 75 (9/12) Tg(MoAPP695. WT) 1859  6 ± 0.8 0.8 ± 0.21 0 (n= ) 0 (n = 2) Tg (MoAPP695.WT) 1869 26 75 (3/4) 100 (4/4) +(1/2) Tg (MoAPP695. WT) 1874 31 ± 3.73.1 ± 0.55 47 (8/17) 79 (11/14) +(3/5) Tg (MoAPP695. WT) 1855 29 2.7 ±0.1  0 (n = 2) 100 (2/2)

Behavioral Analyses

To determine whether FBV mice naturally became behaviorally impairedwith advancing age (the mouse equivalent of senile dementia in humans,or the old dog which has forgotten its tricks), FBV mice were observedup to one year and the behavior of these aged mice compared to that oftransgenic mice. Behavioral analyses were usually performed three timesper week using the corner index (CI) test. The test exploits a strikingneophobic response which occurs in many affected transgenic mice. Theneophobic response is manifested by a decrease in exploratory activityspecific to testing in a novel chamber. Early in the clinical course,affected mice often appear normal in their home cages but exhibittransient immobility for 30 to 60 seconds after being placed alone in aclean cage, in contrast to unaffected mice which typically explore andsniff around the novel setting. A characteristic response of an affectedmouse is to hold its neck low with its tail stiff during the transientimmobility. Alternatively, an affected mouse runs to a corner and thenassumes a crouched or frozen posture there. The (CI) test measures thenumber of times a mouse sniffs the corners of a clean cage during thefirst 30 seconds after it is placed alone into that cage. Based upon thecollective observations of >2000 tests of >100 transgenic mice and >2500tests of >140 non-transgenic mice, we established criteria for thepresence of a behavioral disorder were determined to be scores of two“0's” or “0 and 1” occurring within three consecutive tests. The onsetof illness is ascribed to the first of three consecutive testing datesin which abnormal scores were obtained.

To perform the corner index test, a test mouse, held by the tail, isplaced in the center of a clean cage that is otherwise identical to itshome cage. The number of times the mouse sniffs the corners of the testcage during the first 30 seconds after it was placed into that cage arerecorded as the CI. Animals which are obviously moribund beforeattaining the CI criteria and animals which develop witnessed seizuresalso are diagnosed as ill. Animals housed alone are excluded from theanalysis because several non-transgenic and transgenic mice obtain lowscores while housed alone without displaying the characteristic freezingpostures of the affected transgenic animals. When these mice are housedwith other mice, their CI scores increase. To control the variations indiurnal activity, all animals are tested between 1430 h and 1830 h.

An Age-Related CNS Disorder in FVB Mice

Behavioral Abnormalities.

The life expectancy of FVB mice is approximately 600 days but little isknown about age-related CNS disorders in FVB mice. To determine whetherFVB mice naturally become behaviorally impaired with advancing age, 110FVB mice 150-500 days of age from three different institutions(University of Minnesota, Minneapolis, Minn., McLaughlin ResearchInstitute, Great Fall, Mont., and Harlan Sprague Dawley, Inc.Indianapolis, Ind.) were observed. With advancing age, 18 mice as earlyas 154 days of age developed behavioral abnormalities, includingagitation, inactivity, seizures, and neophobia, as defined by the cornerindex test, and premature death (Table 6). Another six mice died fromtumors or accidentally. Although agitation or inactivity occurred in allaffected transgenic mice, these were subjective signs that rarelyappeared in most normal mice. The onset of illness was defined by cornerindex test results in conjunction with the observation of seizures,agitation or apathy. Both male and female mice were affected. Threeagitated mice died prior to diagnosis by corner index criteria. Onedeath occurred immediately following an observed seizure. The remainingmice grew progressively less active, and were sacrificed for pathologicstudies between nine and 91 days after the onset of abnormal behavioralsigns. The cumulative incidence of behavioral abnormalities and death(excluding accidental and tumor-related deaths) in this cohort of FVBmice was 23% by 500 days of age (see FIG. 9).

Gliosis. Brains from sixteen older non-transgenic FVB mice nine totwelve months of age, seven exhibiting the abnormal behaviorcharacteristic of affected transgenic APP mice and nine age-matchedbehaviorally normal mice, were examined in a coded fashion. Six of theseven brains from the behaviorally abnormal mice exhibited profoundhypertrophic astrocytic gliosis in the hippocampus, parahippocampalarea, amygdala, and cerebral cortex (FIG. 10). None of the brains fromthe nine age-matched, behaviorally normal mice exhibited this degree ofgliosis, although moderate gliosis restricted to the hippocampus wasobserved in some mice. These findings indicate that the behavioraldisorder in affected older non-transgenic mice is tightly associatedwith cortico-limbic gliosis (Yates-corrected X²=8.96, p=0.003). Thebrains of the non-transgenic behaviorally impaired FVB mice showed nonamyloid plaque deposition, neurofibrillary tangle formation, neuronalabnormalities, or qualitative changes in neuronal or glial numbers.

Regional-cerebral glucose utilization. To obtain an independentfunctional assessment of the abnormal behavior observed in impaired FVBmice, regional brain glucose utilization was determined using amodification of the Sokoloff method (Sokoloff, et al., J. Neurochem.28,897-916 (1977)). Regions associated with learning, memory, andemotion such as the cerebral cortex, hippocampus, entorhinal cortex, andamygdala, which are most impaired in cognitively impaired aged humansand patients with AD were examined. Densitometric values of¹⁴C-deoxyglucose distribution were normalized to cerebellar valuesbecause the cerebellum appeared uninvolved clinically andpathologically. The regional cerebral glucose utilization in cerebraltissue in impaired FVB mice was compared to that in cerebral tissue inbehaviorally normal, age-matched FVB mice. Significant decreases(p<0.05, analysis of variance) in regional glucose utilization,particularly in the hippocampus (−42%), amygdala (−43%), entorhinalcortex (−46%), parietal cortex (−34%), frontal cortex (−19%) andtemporal cortex (−18%), were observed in the cerebral tissue in theimpaired FVB mice. In contrast, no significant decreases were observedin several structures, including the corpus callosum, medullaryreticular formation, dentate nucleus, and vermis.

The development of impaired behavior accompanied by cortico-limbichypertrophic gliosis and diminished regional cerebral glucoseutilization, especially in the cerebrum, in FVB mice defines acharacteristic age-related CNS disorder with features of the senescentchanges observed in other rodent species, such as hypertrophic gliosisand diminished regional glucose utilization in limbic and corticalstructures. Although the age-related behavioral abnormalities observedin impaired FVB mice have not been described to occur naturally in otherrodents, the major decrease in regional cerebral glucose utilizationfound in the cortico-limbic areas of the brain involved in learning,memory, and emotion, strongly suggest that some, if not most, of thebehavioral abnormalities in affected FVB mice reflect dysfunction inthese brain regions. Because the behavioral, pathological, andfunctional abnormalities observed in these mice share features found inother aged, impaired rodents and in demented humans, the constellationof findings represents a form of CNS senescence in FVB mice.

Transgenic Mice Expressing Mutant and Wild-Type APP

Behavioral abnormalities. An abnormal phenotype resembling that in aged,impaired FVB mice developed in animals expressing high levels of APP.Copy number per se was unlikely to be the direct cause of the CNSdisorder, since a previously published transgenic line developed in FVBmice, Tg(HuPrP)FVB-152, expressing human PrP driven by 30-50 copies ofthe hamster PrP gene cosmid exhibited no premature behavioralabnormalities or death (Telling, et al., (1994) Proc. Natl. Acad. Sci.U.S.A. 91, 9936-9940). The phenotype in TgAPP mice segregated accordingto the species, genotype and level of APP expression in four linesharboring roughly equivalent copy numbers (20-30: Tg(HuAPP695.WYmyc)466,Tg(MoAPP695.Wtmyc)6209. To determine whether PrP levels were affected bythe presence of supernumerary PrP gene components, brain PrP levels weremeasured in Tg(HuAPP695.TRImyc)1130 mice with 74 transgene copies andnon-transgenic mice. No differences were found, indicating thatalterations in PrP expression were also not the cause of the abnormalphenotype.

Affected transgenic animals developed all the clinical signs observed inaged, impaired non-transgenic FVB mice, including agitation, increasedstartle responses, apathy, and neophobia (Table 6), but they occurredwith significantly high penetrance at earlier ages (FIG. 9, Table 5).Later in the course inactivity and failure to reproduce developed butthere was no tremor, incoordination, weakness, paralysis, or apparentloss of sensation as judged from their withdrawal or vocal responses totail or foot pinching. Seizures were observed in a small percentage (3%(6/181)) of affected Tg(HuAPP695.TRImyc) mice. It is possible that theactual incidence of seizures is higher, and would be detected if micewere observed for more than 30-60 seconds three times per week.

Behavioral abnormalities in transgenic mice developed as early as onemonth of age. There was no significant difference between the onset ofbehavioral abnormalities in male and female mice. Some transgenic mice(<14%) overexpressing APP died as early as one month of age withoutexhibiting prior seizures or neophobia. A neuropathologic examination oftwo of these mice identified cortico-limbic gliosis indistinguishablefrom transgenic mice that had died after exhibiting the characteristicbehavioral signs, so it is probable that these mice died as a result ofthe same disorder as the other affected transgenic mice.

Small stature was observed in animals with transgenic brain APP levelsexceeding twice the endogenous levels (Table 5). This difference in sizewas not apparent at birth but became conspicuous by four to six weeks ofage, and was less or absent in older animals. The transgenic animalsappeared normally proportioned. Small size was not required forbehavioral abnormalities to occur, since Tg(HuAPP695.TRImyc)1118 micedied prematurely and developed behavioral abnormalities despite beingnormal in size.

TABLE 6 Clinical and pathological signs in aged, impaired FVB mice andin affected FVB mice expressing APP transgenes % aged, impaired FVBSigns mice % affected Tg FVB mice Seizures 17% (3/18 3% (6/181)Agitation or inactivity 100% (18/18) 100% (181/181) Neophobia 83%(15/18) 84% (152/181) Early death (excluding 100% (4/4) 100% (82/82)sacrificed mice Cortico-limbic gliosis 86% (6/7) 76% (16/21)

Pathological Analyses of Transgenic Mice

Brains of transgenic mice exhibiting behavioral abnormalities or founddead and age-matched nontransgenic littermates were examined forneuropathologic abnormalities. Brains were immersion fixed or perfusedwith 10% phosphate-buffered formalin or 4% buffered paraformaldehyde,embedded in paraffin, and cut into 5-8 μm sections on a rotarymicrotome. Tissue sections were stained with hematoxylin and eosin,cresyl violet, thioflavin S, or Congo Red stains, or using theBielschowsky silver or TUNEL (Gavrieli, et al., (1992) Journal of CellBiology 119, 493-501) methods.

For immunohistologic studies, paraffin sections were deparaffinized andrehydrated through xylol and graded alcohols. Endogenous peroxidase wasquenched by treatment with 6% hydrogen peroxide in methanol for 10minutes or with 3.0% hydrogen peroxide in methanol (1:5), and rinsed indeionized water or phosphate buffered saline. To enhance APP antigendetection, selected sections were microwave irradiated in water at fullpower for 15 minutes, cooled to room temperature, transferred todeionized water in 0.5 M TBS (pH 7.6), and pretreated with 0.4% TX/TBS,followed by 3% normal goat serum in TBS. Primary antibodies 6E10 (1:100)and 8E5 (1:100 ascites fluid) were prepared in 0.1% TX/TBS with 2%normal goat serum.

Following incubation for 24 hours, slides were rinsed, incubated ingoat-antirabbit or -antimouse IgG (1:20) in 0.1% TX/TBS, and rinsed inTBS followed by one-hour incubation in rabbit or mouseperoxidase-antiperoxidase (1:100) at room temperature. Rinsed slideswere reacted in the presence of 0.05% diaminobenzidine in 0.01% hydrogenperoxide, rinsed three times in TBS, dehydrated through a graded seriesof alcohols to xylene. Representative sections were silver-enhancedaccording to the Fontana-Masson method (Masson (1928) Am. J. Path.H:118-211), and viewed under transmitted light microscopy anddifferential interference contrast optics. Other sections were immersedin 70% formic acid for 10 minutes, rinsed in phosphate buffered saline,and immersed in 10% normal hose serum for 1 hour. Primary antibody 6E10(1:5000) was prepared in phosphate buffered saline. Following incubationovernight at 4° C., sections were rinsed in phosphate buffered saline,incubated with antimouse IgG, followed by avidin-biotin complex (VectorLabs, Inc.). Rinsed slides were reacted with diaminobenzidine andcounterstained with Harris hematoxylin. GFAP was detected using amonoclonal antibody to GFAP at a dilution of 1:60 in phosphate bufferedsaline.

Gliosis. Using coded specimens, brains from 21 affected transgenic miceexpressing the triple HuAPP variant, the Dutch HuAPP variant, theSwedish HuAPP variant, wild-type HuAPP, as well as brains from 12age-matched, unaffected non-transgenic mice were examined. Brains from16 affected transgenic mice exhibited prominent hypertrophic astrocyticgliosis located predominantly in the parahippocampal area, hippocampus,amygdala, and cerebral cortex (FIG. 10), with relative sparing of thebasal ganglia. The astrocytes had enlarged, elongated processes whenimmunostained for glial fibrillary acid protein (GFAP), but there was noincrease in the number of astrocytes. Brains from the age-matchednon-transgenic mice were devoid of the reactive gliosis, indicating astrong association between gliosis and abnormal behavior(Yates-corrected X²=14.83, p=0.00012). Bielschowsky silver stainsrevealed no neurofibrillary tangles, dystrophic neurites, or neuriticplaques. Neurons appeared normal with Nissl and hematoxylin and eosinstains.

Gross and microscopic examinations of six transgenic mice found deadrevealed characteristic brain pathology (astrocytic gliosis in thehippocampus, cerebral cortex, amygdala, and parahippocampal area, asdescribed below), but no evidence of microscopic or gross pathologyoutside the CNS. Amyloid was specifically excluded by thioflavin Sstaining in the heart, ling, liver, spleen, thymus, kidney, smallintestine, and testes in four of these transgenic mice. The absence ofpathologic findings outside the CNS indicates that the deaths were mostlikely due to causes which were neurologic in origin.

Regional Cerebral Glucose Utilization

To determine whether there were functional differences in the brains ofaffected transgenic mice, regional brain glucose utilization wascompared among affected transgenic mice with aged, impairednon-transgenic FVB mice and age-matched non-transgenic mice. Compared tonormal, non-transgenic littermates, significant reductions (P<0.05;analysis of variance) in glucose utilization were observed in variousforebrain regions in transgenic mice, including the hippocampus (−31%),amygdala (−28%), parietal cortex (−34%), temporal cortex (−33%), andoccipital cortex (−36%). Many regions, in contrast, showed nosignificant reduction (p>0.05), including the sensory-motor cortex,corpus callosum, reticular formation, vermis, vestibular complex, anddentate nucleus. The diminution of regional glucose utilization wasparticularly pronounced in the hippocampus, amygdala, and some corticalregions in affected transgenic mice closely resembling that occurring inolder, impaired non-transgenic FVB mice.

Extracellular Aβ Staining in a Transgenic Mouse

One animal shows extracellular staining with an antibody described inSaido, et al., J. Biol. Chemistry 269:15253-15257 (1994). This antibodyspecifically stains the aminoterminus of Aβ. It is an affinity purifiedpolyclonal antibody. The staining in our transgenic mouse can be blockedby specific competition with the Aβ fragment. The staining pattern inour transgenic mouse resembles that which is seen in AD brain stainedwith the same antibody. More animals are being examined. Furthercharacterization with other antibodies is being done. Ultrastructuralstudies also being done.

Example 7 Expression of APP Transgenes in FVB/N Mice

Transgene Construction

The PrP-APP transgenes were generated by inserting Sal1-flanked human ormouse APP ORFs into a hamster PrP cosmid vector. This vector is a ˜40 kbfragment of genomic DNA containing the hamster PrP gene with ˜20 kb ofupstream sequences, in which the hamster PrP ORF is replaced by a uniqueSal1 restriction site. The HuAPP695.SWE, HuAPP695.TRImyc, andHuAPP695.TRImyc, and APP sequences were modified for strong translationinitiation. The 5′ end of the APP coding sequence is preceded by a Sal1site and a strong Kozak translation initiation sequence(5′-GTCGACACC-ATGCTGCCC. . . (SEQ ID NO:16)), and the 3′ end of the APPcoding sequence is immediately followed by a Sal1 site (. ..AACTAGCAGCTG-3′ (SEQ ID NO:17)); start and stop codons are underlines;site in boldface). These modifications and the APP mutations were madeusing standard cloning methods and polymerase chain reaction(PCR)-based, site-directed mutagenesis. The PrP-APP cosmids weredigested with Not1, which releases the PrP-APP fusion gene from thepcos6EMBL vector. The PrP-APP fusion genes were isolated after sizefractionation on an agarose gel and electroeluted. The PrP-APP fusiongene was further purified with organic solvents and precipitated inammonium acetate and ethanol. The PrP-APP fusion genes were dissolved in5 mM Tris-Cl (pH 7.4) or 10 mM Tris-Cl (pH 8.0) to a final concentrationof 2-2 μg/ml prior to embryo injection.

Transgenic Mouse Generation and Screening

Transgenic lines were initiated by microinjection of single-cell mouseembryos. The embryo donors and fertile studs were inbred FVB/N miceobtained from the National Cancer Institute (NIH). Post-weaning tailbiopsy DNA was generated and 1 μl of unpurified DNA was used in a 25 μlPCR reaction. To detect PrP-APP fusion DNA, the PrP-APP fusion DNA wasamplified using the PCR with a pair of oligomer primers, 1503:(5′-CTGACCACTCGA-CCAGGTTCTGGGT-3′ and 1502(5′GTGGATAACCCCTCCCCCAGCCTAGACCA-3′), located in the 3′ region of APPand the 3′ untranslated region of PrP, respectively (See FIG. 8). The1503 primer recognizes a region that is homologous in mouse and humanAPP and could therefore be used to detect both PrP-MoAPP and PrP-HuAPPDNA. Using primers 1502 and 1501 (5′-AAGCGGCCA-AAGCCTGGAGGGTGGAACA-3′),a parallel PCR reaction applying a fragment of murine PrP was performedas a positive control.

Transgene copy number analysis was performed using 5 μg of denaturedpurified tail DNA baked onto nitrocellulose and hydridized to aradiolabeled 1.3 kb Sal1-Xho1 DNA fragment encoding a segment of thehamster PrP 3′ untranslated region located in the hamster PrP cosmidvector. After two high stringency washes, the relative intensities ofsignals from genomic DNAs of transgenic mice and hamsters were comparedusing a phosphorimager to obtain transgene copy numbers relative todiploid hamster genomic DNA.

Analysis of Transgene Expression

APP transgene products were examined in progeny of transgenic founderssacrificed at 1-4 months of age. Quantitative immunoblotting of extractsfrom brain homogenates was carried out in parallel with extractsprepared from age-matched nontransgenic littermates. Homogenates (20%,w/v) of brain tissues were prepared in TNE (50 mM Tris-Cl(pH 8.0), 150mM NaCl, 5 mM EDTA with 2% phenylmethylsulfonyl fluoride) buffer using ahand-held polytron. Homogenates were diluted with an equal volume ofTNE, 1% Nonidet P-40, 1% deoxycholate, 0.4% SDS and sonicated in a bathsonicator until all viscosity was lost. Homogenates were then boiled for10 min. and centrifuged at 10,000×g for 10 min. the supernatants weremixed with an equal volume of 2× sample buffer (Laemmli, 1970), boiled 2min. and fractionated using a 6% SDS-polyacrylamide gel. Proteins wereelectrophoretically transferred to Immobilon membranes (Pierce) andincubated with monoclonal (22C11 and 6E10) anti-APP antibodies. Reactivemonoclonal antibodies were visualized following incubation withsecondary rabbit antibodies to mouse IgG before incubation with¹²⁵I-protein A. Radioactivity was quantified on a phosphorimager(Molecular Dynamics, Inc.).

Analysis of Aβ in Brain Tissue

Approximately 0.2 g of tissue was dounce homogenized (4 strokes) in 1 mlof 70% glass-distilled formic acid. Homogenates were centrifugedat >100,000×g for 1 hr. The formic acid extract (layered between anoverlaying lipid layer and a small pellet) was removed, and a smallaliquot was diluted 50 times in 1 M Tris (pH 8.0). This sample was thenfurther diluted 2.4 times in Buffer EC (0.02 M sodium phosphate (pH7.0), 0.2 mM EDTA, 0.4 M NaCl, 0.2% bovine serum albumin, 0.05% CHAPS,0.4% Block-Ace, 0.05% sodium azide), and 100 μl of this was analyzeddirectly using either the Ban50/Ba27 or Ban50Bc05 ELISA systemsdescribed previously (Suzuki et al., 1994; Gravina et al., 1995). Aβvalues reported were obtained by comparing the absorbance obtained fromduplicate samples to standard curves of either Aβ₁₋₄₀ (Ban50/Ba27) orAβ₁₋₄₂ (Ban50/Bc05) obtained from Beachem. These values were correctedfor dilution and initial wet weight of the tissue and are expressed aspicomoles per gram of wet weight. All samples were coded with respect tothe transgenic status of the animals.

Behavioral Analyses: Neophobia:

To perform the corner index test, a test mouse held by the tail isplaced in the center of a cage (18×30×13 cm) with clean bedding (soiledbedding removed between tests), and the number of times the mouse sniffsthe corners of the test cage during the first 30 s after being placedinto the cage is recorded as the corner index (Cl). (See Specification,page 18, lines 1-18). Animals are usually tested 3 times per week. Lowscores in animals housed alone were excluded from the analysis unlessthey displayed thigmotaxis or the characteristic freezing posture ofother neophobic transgenic mice. To control for variations in diurnalactivity, all animals were tested between 1300 hr and 1830 hr. Criteriafor the presence of neophobia in non-transgenic mice >150 days of agewas ≧3 consecutive scores of 0.

Pathological Analyses of Mice

Brains of mice exhibiting behavioral abnormalities or found dead andage-matched littermates were examined for neuropathologic abnormalities.Brains were immersion fixed or perfused with 10% phosphate-bufferedformalin or 4% buffered paraformaldehyde, embedded in parafin, and cutinto 5-8 μm sections. Tissue sections were strained with hematoxylin andeosin, cresyl violet, thioflavin S, or Congo red stains, or by using theBielschowsky silver methods.

For immunohistologic studies, endogenous peroxidase was quenched bytreatment with 6% hydrogen peroxide in methanol or with 3.0% hydrogenperoxide in methanol (1:5). To enhance APP antigen detection, selectedsections were microwave-irradiated in water at full power for 15 min.cooled to room temperature, transferred to deionized water in 0.5 M TBS(pH 7.6), and pretreated with 0.4% Triton X-100 in TBS (TX/TBS),followed by 3% normal goat serum in TBS. Primary antibodies 6E10 (1:100)and 8E5 (1:100 ascites fluid) were prepared in 0.1% TX/TBS with 2%normal goat serum. Following incubation for 24 hr., slides wereincubated in goat anti-rabbit or anti-mouse IgG (1:20) in 0.1% TX/TBS,followed by a 1 hr. incubation in rabbit or mouseperoxidase-antiperoxidase (1:100) at room temperature. Rinsed slideswere reacted in the presence of 0.05% diaminobenzidiine in 0.01%hydrogen peroxide. Representative sections were silver enhancedaccording to the Fontata-Masson method (Masson, 1928). Other sectionswere immersed in 70% formic acid for 10 min., rinsed in PBS, andimmersed in 10% normal horse serum for 1 hr. Following incubationovernight at 4° C. with primary antibody 6E10 (1:5000), sections wererinsed in PBS and incubated with anti-mouse IgG, followed byavidin-biotin complex (VectorLabs, Inc.). Rinsed slides were reactedwith diaminobenzidine and counterstained with Harris hematoxylin. GAFPwas detected using a monoclonal antibody to porcine GFAP (Sigma).

Regional Brain Glucose Utilization Analysis

Mice received an intraperitoneal injection of (¹⁴C)2-deoxyglucose (NewEngland Nuclear; 5 μCi in 0.4 ml of 0.9% NaCl) and were sacrificed 60min. later. Brains were rapidly removed and frozen in isopentane cooledto −30° C. with dry ice. A sample of trunk blood was collected and usedfor determination of plasma glucose concentration by a glucose analyzer(Beckman). Techniques for quantitative autoradiography were according tothe methods described by Ladecola et al., 1983; Ladecola and Xu, 1994and are only summarized here. Coronal brain sections (20 μm) were cut ona cryostat (Hacker-Bright), mounted on glass slides, and exposed toX-ray film (Dupont) together with calibrated ¹⁴C standards (Ladecola etal., 1983). The film was developed 10 days later using an automaticdeveloper (Kodak), and the optical density (OD) of regions of interestwas determined bilaterally on four adjacent sections using acomputerized image analyzer (MCID system. Imaging Research Inc.). OD wastransformed into ¹⁴C concentration (nCi/g) using the standards on thefilm. Owing to the small size of some mice (15-20 g), blood sampling fordetermination of the 2-deoxyglucose arterial time course could not beperformed, except at the time of sacrifice. Therefore, a CGU index wasobtained by dividing regional radioactivity values (nCi/100 g/min) bythe radioactivity of a region devoid of pathology, the whole cerebellum.This normalization procedure has been validated and widely used in smalllaboratory animals (e.g., Sharp et al., 1983; Mitchell and Crossman,1984; Williot et al., 1988). In our experiments, the rate of ¹⁴ Caccumulation in cerebellum (nCi/100 g/min) and plasma glucose did notdiffer between control, aged, and transgenic mice. This findingindicates that the CGU index provides an accurate estimate of glucoseutilization as determined by the method of Sokoloff et al. (1977).

PrP Cosmid Vector Drives Overexpression of APP in Transgenic Mice

To determine the effect of mutant and wild-type APP expression FVB/Nmice, we replaced the prion protein (PrP) open reading frame (ORF) witha variety of APP ORFs in a hamster PrP cosmid vector. Transgenic miceharbored one of four different transgenes, some containing mutationsassociated with familial AD (MoAPP695.WT (wild type); HuAPP695.SWE(K670N and M671L, APP₇₇₀ numbering); HuAPP695.TRImyc (V7171, V721A, andM722V with a 3′-myc tag); HuAPP695.WTmyc (wild type with a 3′-myc tag);Mo, Mouse; Hu, Human). Initially, we introduced transgenes with a 3′ myctag, a 12 codon segment of the c-myc proto-oncogene, to facilitateimmunodetection of transgene products (Wong and Cleveland, 1990). Themyc tag exerted no apparent effect on the phenotype, sinceTg(HuAPP695.SWE) mice lacking the myc tag developed the same clinicaland pathologic features and those with the myc tag; the high level ofAPP expression obtained in our mice obviated the need for the myc tag.The experimental V721A and M722V mutations, unintentionally introducedto the APP ORF harboring the V7171 mutation linked to early onsetfamilial AD and discovered after transgenic lines had been established,exerted no obvious effect on the phenotype since Tg(HuAPP695.TRImyc)mice developed the same clinical and pathologic abnormalities astransgenic mice expressing the other three transgenes. Subsequentanalyses of HuAPP695.TRImyc in cultured cells indicated that theseunintentional mutations exert no significant effects on the processingof HuAPP relative to protein maturation, modification, or proteolyticprocessing to produce soluble actodomains or Aβ peptides.

APP expression was measured in brains of transgenic mice harboringdifferent transgene copy numbers by quantitation of immunoblots intransgenic lines with the monoclonal antibody 22C11 which recognizes anidentical epitope in both mouse and human APP as well as amyloidprecursor-like protein 2 (APLP2), potentially leading to anunderestimation of the amount of transgenic APP relative to endogenousMoAPP. APP protein expression in transgenic brain depended upon copynumber as well as the species of APP expressed: MoAPP transgenesachieved levels equivalent to those of HuAPP transgenes, but with fewercopies.

Measurement of Aβ in Tg(HuAPP695.TRImyc) mice indicates that both Aβ₁₋₄₀and Aβ₁₋₄₂ are generated in the brain. Aβ levels were not measured intransgenic FVB/N mice expressing HuAPP.SWE because of insufficientnumbers of mice, owing to their poor breeding characteristics, and Aβlevels were not measured in transgenic mice expressing MoAPP becausemethods for reliably measuring mouse Aβ in the brain are not yetavailable. The Ban50 capture antibody does not recognize MoAPP; levelsindicated for non-transgenic mice represent background signal. Bothforms of Aβ were readily detectable in transgenic mice but weresignificantly higher in lines overexpressing APP and exhibiting clinicalabnormalities than in an unaffected line expressing lower levels of APP.

Specific immunostaining for human APP/Aβ using the 8E5 or 6E10monoclonal antibodies revealed HuAPP in vesicular structures withinlarge pyramidal cells of the hippocampus, parahippocampal area,amygdala, and cerebral cortex, as well as fainter staining throughoutthe brain in smaller neurons and some glial cells. Antibody 8E5 (gift ofDale Schenk, Athena Neurosciences) recognizes a segment of APP spanningresidues 519-667 (APP₇₇₀ numbering), and 6E10 recognizes residues 1-17of human Aβ (Kim et al., 1990). The pattern of HuAPP immunostainingmatched that of regional brain immunoblots which showed the highestlevels of expression in the cerebrum. The brain and spinal cordcontained the highest levels of HuAPP; the striated muscle, heart, skin,and lung contained <5% of brain levels; in the thymus, liver, spleen,kidney, testes, and small intestine HuAPP was undetectable.

PrP levels remained unchanged in animals with high transgene copynumbers, indicating that the PrP promoter and other sequences in thetransgenes did not deplete transcription factor pools, and that thecellular machinery for synthesizing, modifying, and translocatingmembrane glycoproteins was not overburdened.

Behavioral Abnormalities: Neophobia and Other Neurologic Signs

The corner index test revealed a striking difference between transgenicand non-transgenic mice. Corner index scores for non-transgenic miceshowed few values ≦1 during the first 3 months, while scores of sometransgenic mice overexpressing APP showed values ≦1 with advancing age.The low scores appear to reflect a neophobic response. Based on >2000tests of >100 transgenic mice and >2500 test of >140 non-transgenic mice<150 days of age, the age when two scores of “0” or a “0” and “1”appeared within three consecutive testing sessions defined the onset ofneophobia. None of the 100 non-transgenic mice tested through 100 daysof age or of the 48 non-transgenic mice tested through 150 days of agefailed the corner index test. Neophobia developed as early as 1 month ofage in both male and female transgenic mice overexpressing APP andpreceded death by an average of 40 days in transgenic1130H mice. Sixtransgenic FVB/N lines and 4 additional founders expressing high levelsof wild-type MoAPP695.WT, HuAPP695.SWE, HuAPP695.WTmyc, orHuAPP695.TRImyc exhibited neophobia. Mice failing the corner index testalso exhibited other neurologic signs, including thigmotaxis, agitation,still tail, stare, tremulousness, and inactivity. Of 181 mice fromaffected lines, 6 had generalized tonic-clonic seizures during cornerindex testing.

We also generated transgenic FVB/N mice overexpressing wild-type MoAPP;37% of transgenic1855 and 54% transgenic1874 mice were neophobic at 100days, and 11% of transgenic1874 mice were dead at 100 days. The rate ofdevelopment of neophobia was lower in transgenic mice expressingMoAPP695.WT than in transgenic mice expressing HuAPP695.TRImyc.

Regional Cerebral Glucose Utilization

To identify the affected areas of the brain in neophobic transgenic andenophobic mid- to late-adult non-transgenic FVB/N mice, regional brainglucose utilization was determined by densitometric measures of(¹⁴C)deoxyglucose levels (μCi/100 g/min). Regional cerebral glucoseutilization in neophobic Tg1130H and age-matched non-transgenic mice wascompared. The former exhibited significant reductions in glucoseutilization in various cortico-limbic regions, including the entorhinalcortex (−37%; p=.008), hippocampus (−30%; p≦.003), and amygdala (−28%;p=.004) as well as the parietal (−34%; p=.001, temporal (−33%; p=.017),and occipital (−36%; p=.001) lobes of the cerebral cortex. Thesomatosensory-motor cortex was relatively spared, corroborating theapparent absence of motor and sensory abnormalities in these mice, andmany brain stem regions, including the pontine reticular formation,vestibular nuclear complex, and dentate nucleus, showed no significantreduction in glucose utilization.

Astrogliosis Without Amyloid formation in Brains of Transgenic FVB/NMice

Using coded specimens, we examined brains of 19 neophobic transgenicmice expressing HuAPP695.SWE, HuAPP695.WTmyc, HuAPP695.TRImyc, orMoAPP695.WT as well as 12 age-matched, unaffected non-transgenic mice(see Table 2). Fifteen brains from affected transgenic mice exhibitedprominent hypertrophic astrocytes located predominantly in theparahippocampal area, hippocampus, amygdala, and cerebral cortex, withrelative sparing of the basal ganglia. The astrocytes had enlarged,elongated processes when immunostained for glial fibrillary acidicprotein (GFAP), and there was no apparent increase in the number ofastrocytes. Brains of ace-matched non-transgenic mice were devoid ofreactive gliosis. In general, there was an association between gliosisand abnormal behavior (Yates-corrected X²=14.83, p=.00012). Bielschowskysilver stains revealed no neurofibrillary tangels, dystrophic neurites,or neuritic plaques. Neurons appeared normal with Nissl and hematoxylinand eosin stains.

Seven non-transgenic FVB/N mice 9-12 months of age exhibiting neophobiaand 9 age-matched, behaviorally normal mice were examined in a codedfashion. Six of the 7 brains from neophobic mice exhibited pronouncedastrocytic gliosis in the hippocampus, parahippocampal area, amygdala,and cerebral cortex as detected by GFAP staining. The neostriatum showedlittle or no astrocytosis. None of the brains from the 9 age-matched,behaviorally normal mice exhibited this degree of gliosis, althoughmodest gliosis restricted to the hippocampus was observed in somecontrol FVB/N mice. These findings indicate that neophobia innon-transgenic FVB/N mice is associated with gliosis in the cerebralcortex and limbic brain regions (Yates-corrected X²=8.96, p=.003). Thebrains of these mice showed no amyloid deposition, neurofibrillarytangles, neuronal abnormalities, or qualitative changes in neuronal orglial numbers. To detect APP or Aβ immunoreactivity in brain tissue fromanimals with clinical abnormalities in transgenic FVB/N linesoverexpressing HuAPP, we used two antibodies: 8E5 antibody, whichstained amyloid and intraneuronal vesicular structures in microwavedtissue sections from patients with AD, and 6E10 antibody, which stainsamyloid from patients with AD only after formic acid pretreatment ofbrain tissue. In 4 Tg(HuAPP695.SWE) mice and 7 Tg(HuAPP695.TRImyc) mice,neither the microwave nor formic acid pretreatment of brain tissuerevealed extracellular APP or Aβ immunoreactivity using theseantibodies. Amyloid deposits were not demonstrable by staining withCongo red or thioflavin S. We concluded that the abnormal phenotype inthese transgenic mice occurred independently of amyloid plaquedeposition.

The distinction between age-dependent penetrance of death and neophobiafor FVB/N mice expressing MoAPP and HuAPP transgenes indicates that APPtransgenes with different amino acid sequences differ in theirage-dependent potency as regards the effect. However, the qualitativefeatures of the phenotype we observe in all transgenic FVB/N miceoverexpressing APP resemble an acceleration of a naturally occurring CNSdisorder in FVB/N mice, regardless of the primary structure of APP.Although it is possible that the presence of two additionaltransmembrane mutations in HuAPP.TRImyc could diminish generation of Aβ,and thereby alter the phenotype, our date indicate that mice expressingthis transgene are in fact able to generate both Aβ₁₋₄₀ and Aβ₁₋₄₂ anddevelop the same clinical abnormalities as transgenic mice expressingHuAPP695.SWE, HuAPP695.WTmyc, and MoAPP695.WT transgenes.

Example 8 Correlative Memory Deficits, Aβ Elevation and Amyloid Plaquesin Transgenic Mice

Tg(HuAPP695.K670N-M671L)2576 mice were generated by driving expressionof human βAPP-695 containing K670N-M671L (βAPP-770 numbering), amutation found in a large Swedish family with early onset AD (Mullan, etal., Nature Genetice 1:345-347 (1992)), with a hamster prion protein(PrP) cosmid vector (Scott, et al., Protein Sci 1:986-97 (1992)) inwhich the PrP open reading frame (ORF) was replaced with the variantβAPP ORF (FIG. 15a). Tg 2576 mice produced 5.56±0.33 units (mean±SEM)(73 day-old mice) to 5.76±0.74 units (430 day-old mice) of transgenicbrain βAPP expression, where one unit of expression is equivalent to theamount of endogenous mouse βAPP in non-transgenic littermates (FIG.15b). Transgenic βAPP expression appeared to remain unchanged betweentwo and 14 months of age.

Two groups of seven to nine transgene positive mice and 10 to 11transgene negative littermates underwent spatial alternation testing ina Y-maze at three and 10 months of age. Three groups of nine to 13transgene positive mice and 10 to 14 transgene negative littermatesunderwent spatial reference learning and memory testing in the Morriswater maze (Morris, J. Neurosci. Meth. 11:47 (1984)) at two, six, andnine months of age. The test experience for each set of animals wasnovel, and all animals were tested in a coded manner. The nine to 10month-old animals were N1-generation mice (C57B6×C57B6/SJL F1). The twoto three and six month-old animals were N2-generation mice(C57B6×C57B6/SJL F1). A subset of the N2-generation mice (eighttransgene positive and 10 transgene negative mice) were retested at 12to 15 months of age.

When transgene positive and transgene negative mice were given a choiceof entering either of two arms in a Y-maze, they tended to alternatetheir choices spontaneously. Ten month-old transgene positive mice,however, showed significantly less tendency (p<.03) than age-matchedtransgene negative mice to alternate arms on successive choices (FIG.16a). The behavior of the old transgene positive mice on the spatialalternation task is characteristic of animals with damage to thehippocampal formation (Douglas, Spontaneous Alternation Behavior Richmanand Richman, Eds. (Springer-Verlag, New York, 1990) pp. 73-109).

In another important learning test nine month-old transgene positivemice were impaired in their performance in the water maze relative toage-matched transgene negative mice (FIG. 16b). The water maze testdescribed by Morris (1984) J. Neurosci. Meth. 11:47 was modified for usewith mice. The water maze was a circular pool 1 meter in diameter filledwith water maintained at 20° C. and made opaque by the addition ofpowdered milk. Animals were pretrained by swimming to a 12.7 cm squarePlexiglas platform that was submerged 1.5 cm beneath the surface of thewater and placed at random locations within the pool. Duringpretraining, heavy curtains were drawn around the pool so that mice wereunfamiliar with the extramaze room cues on the first day of spatialtraining. Spatial training consisted of four trials per day, each triallasting until the animal reached the platform or 60 seconds, whichevercame first. After each trial, mice remained on the platform for 30seconds. 24 hours after the 12^(th) and 24^(th) trials, all animals weresubjected to a probe trial in which they swam for 60 seconds in the poolwith the platform removed. Animals were monitored by a camera mounted inthe ceiling directly above the pool, and all trials were stored onvideotape for subsequent analysis of platform crossings and percent timespent in each quadrant during probe trials. Visible platform trainingwas given at least 24 hours following the second probe trial, andconsisted of swimming mice in the same pool described earlier exceptthat the platform was now black, slightly larger (14.2 cm square), andraised above the surface of the water. The platform location was variedrandomly from trial to trial to eliminate the potentially confoundingcontribution of extramaze spatial cues.

In both visible platform and hidden platform versions, animals wereplaced in the pool facing towards the wall of the pool in one of sevenrandomly selected locations Transgene positive mice trained and testedat two or six months of age were not different from age-matchedtransgene negative mice on most measures. The amount of time taken bythe mice to reach the hidden platform (the escape latency) did notdiffer between two month-old transgene positive and negative animals atany point during training, while the latency was significantly different(p<0.05) on every day for nine month-old animals. Six month-oldtransgene positive animals differed from controls in escape latency onlyon the last day of training. Probe trials, in which animals swam in thepool for 60 seconds with the platform removed, were given 24 hours afterthe 12^(th) and 24^(th) trials, and the number of times the animalscrossed the platform location were recorded. This procedure often givesa more precise measure of the animals' knowledge of the platformlocation, and is less confounded by performance factors such as swimspeed. Nine month-old transgene positive mice were significantlydifferent (p<0.05) from age-matched transgene negative mice on thesecond probe trial, while two month-old and six month-old animals showedno differences on either probe trial.

When 12 to 15 month-old N2 generation transgene positive mice wereretested in the water maze (after rearranging the extramaze cues), theyshowed significantly impaired performance compared to transgene negativelittermates on escape latencies after the 5^(th) trial block and onprobe trials given after the 6^(th) and 9^(th) trial blocks (FIG. 16c).These data suggest that the age-related learning impairment seen in N1generation transgene postive mice can occur despite further geneticdilution of the SJL strain (FIG. 16c). Note that although the escapelatencies of the transgene positive N2 mice are significantly longerthan their transgene negative littermates, they are also shorter thannaïve animals of comparable age. Thus deficits in escape latency in agedtransgene positive animals are unlikely to result from difficulty inswimming, since aged mice given sufficient practice can swim as well asyounger mice.

Since it is possible that the performance of older transgene positivemice is attributable to sensory or motor impairments, we also testednine month-old mice on the visible-platform version of the water maze(FIG. 16d). Although differences in escape latency were evident on thesecond and fourth of four training days, there were no differences onDay 1. These data suggest that while older transgene positive mice mayshow generalized cognitive impairment, they are capable of performing aswell as controls when both are relatively naïve. We also compared motorperformance of the transgene positive and transgene negative ninemonth-old mice by scoring the total number of times during the probetrial each animal crossed imaginary platforms located in each of thefour quadrants. If impaired animals swim normally but in a randompattern during probe trials, they should cross the center of all fourquadrants combined as many times as unimpaired animals; they will simplycross the target platform fewer times. If, on the other hand, they areimpaired on probe trials simply because they are not swimming, therewill be fewer total platform crossings. In fact, the total number ofplatform crossings for transgene positive (24.4±8.7: mean±SEM) andtransgene negative (29.5±1.4) mice was not significantly differentindicating that motor impairment was not a cause of poor performance inthe water maze.

Following behavioral testing a subset of each group of mice wassacrificed. One hemi-brain was frozen for cerebral cortical Aβmeasurements and the other hemi-brain was immersion fixed forhistopathological analysis. All brains were analyzed in a coded fashion.Measurements of Aβ1-40 and Aβ1-42(43) using either the Ban-50/Ba-27 orBan-50/Bc-05 ELISA systems described previously (Suzuki, et al., Science264:1335-1340 (1994); Gravina, et al., Journal of Biological Chemistry270:7013-7016 (1995)) showed a five-fold increase in Aβ1-40 levels(p=0.03, rank sum test) and a 14-fold increase in Aβ1-42(43) levels(p=0.03, rank sum test) between the youngest (two to eight month) andoldest (11 to 13 month) transgene positive animals (Table 7). Thus therewas a good correlation between significantly elevated Aβ levels and theappearance of memory and learning deficits in the oldest group oftransgene positive animals.

TABLE 7 Age Amyloid when plaques Transgene sacrificed Aβ(1-40) Aβ(1-42)4G8*, Mouse # status (days) pmol/gm pmol/gm 6E10⁵⁵⁴ Mice sacrificedbetween 11-13 months of age; transgene positive mice showed impairedspatial alternation and reference memory A01484 Positive 361 325 219 +++A01488 Positive 354 192 129 ++ A01489 Negative 354 <2 <2 ± A01492Negative 371 <2 <2 − A01493 Positive 368 273 177 ++++ A01495 Negative354 <2 <2 − A01496 Negative 354 <2 <2 ± Mean ± SEM Aβ levels in 264 ± 38175 ± 26 transgene positive mice: Mice sacrificed between 6-8 months ofage; transgene positive mice showed no learning and memory impairmentA01984 Negative 233 <2 <2 ± A01987 Negative 219 <2 <2 − A01989 Positive219 45 18 − A02561 Negative 214 <2 <2 A02595 Negative 207 <2 <2 Micesacrificed between 2-5 months of age; transgene positive mice showed nolearning and memory impairment A02428 Negative 139 <2 <2 − A02429Negative 139 <2 <2 A02430 Negative 139 <2 <2 − A02565 Positive 118 71 21A02900 Negative 85 <2 <2 A03103 Positive 67 32 2 A03107 Positive 67 4510 Mean ± SEM Aβ levels in 48 ± 8 13 ± 4 transgene positive mice: *Braintissue was stained with 4G8 (Kim, et al., Neurosci. Res. Commun.2:121-130 (1988)), which recognizes both mouse and human Aβ. ^(†)Allamyloid deposits stained with 6E10 (Kim, et al., Neurosci. Res. Commun.7:113-122 (1990)), which specifically recognizes human Aβ. Noextracellular 6E10 staining was detected in three 105 to 106 day-oldtransgene positive mice or one 155 day-old transgene positive mouse(A01480, A01547, A01548, and Tg2576 founder - not included in behavioralstudies shown).

Aβ deposits were immunoreactive with antibodies recognizing β(1-5)(Saido, et al., J. Biol. Chemistry 269:15253-15257 (1994)), β(1-17)(Kim, et al., Neuroscience Research Communications 7:113-122 (1990)),β(17-24) (Kim, et al., Neurosci. Res. Commun. 2:121-130 (1988)),β(34-40) (Mak, et al., Brain Research 667:138-142 (1994)), β42/43 (Yang,et al., Neuroreport 5:2117-2120 (1994)) and free β42 (Harigaya, et al.,BBRC 211:1015-1022 (1995)). The same plaques were readily identifiedwith multiple antibodies on adjacent sections and were not seen withpreimmune or non-specific ascites and the immunoreactivity waseliminated by preabsorption with the relevant peptides (FIG. 17).Deposits could not be found in the older transgene negative or youngertransgene positive or negative mice examined. Both classic senileplaques with dense amyloid cores and diffuse deposits were present. Thedeposits were found in frontal, temporal and entorhinal cortex,hippocampus, presubiculum, subiculum and cerebellum in all three micewith elevated Aβ by ELISA assay. Dense amyloid plaques were mostfrequent in cortex, subiculum and presubiculum. The dense amyloiddeposits were readily detected with thioflavin S fluorescence andtypically also labeled with Congo red giving the characteristic applegreen birefrigence of classical amyloid (Puchtler, et al., J. Histochem.Cytochem. 10:355-363 (1962)). Some small deposits had the “Maltesecross” signature pattern of the amyloid cores found in AD brain. Underhigh magnification, the thioflavin S and Congo red positive amyloidplaques usually exhibited wisps or fibers radiating from the centralmass which was often ringed by glial nuclei with both astrocytic andmicroglial morphology. GFAP immunoreactive astrocytes were associatedwith amyloid deposition. Staining by the Gallyas silver method revealeddystrophic neurites surrounding dense core plaques.

In contrast to sporadic AD brain, antibodies to β1 and both free β42 andβ(34-40) (which preferentially recognizes x-40) labeled the majority ofdeposits. This may reflect the βAPP670/671 mutations which greatlyincreases cleavage at the β1 site leading to high levels of allfragments beginning with the β1 epitope in contrast to the 717 mutationswhich increase the percentage of x-42 (Suzuki; et al., Science264:1335-1340 (1994); Citron, et al., Nature 360:672-4 (1992)).

Our results demonstrate the feasibility of creating transgenic mice withboth robust behavioral and pathological features resembling those foundin AD, Tg2576 mice younger than nine months of age showing nosignificant deficits in spatial reference or spatial alternationlearning and memory tasks possessed moderate levels of Aβ and no amyloidplaques. Impairment in learning and memory became apparent in mice ninemonths of age and older, correlating with markedly increased levels ofAβ and accompanied by numerous amyloid plaques and Aβ deposits. The risein Aβ levels cannot be explained by a rise in transgenic βAPPexpression, which appeared to remain unchanged with age. Aβ1-42(43)levels rose more dramatically than Aβ1-40 levels. Interestingly, thisparallels the finding in humans with presenilin 1 and presenilin 2mutations exhibiting more significant elevations of Aβ1-42(43) thanAβ1-40 in serum and cultured fibroblasts (S. Younkin, unpublished data).Ongoing studies correlating individual performance in learning andmemory tests with levels of Aβ and extent of amyloid deposition arebeing done to ascertain the contribution of each parameter to behavioraldeficits.

Earlier attempts to produce transgenic mice with robust extracellular Aβdeposits were largely unsuccessful with the exception of mice reportedby Games and colleagues (Games, et al., Nature 373:523-527 (1995)). βAPPexpression was driven in their mice by a PDGF promoter. Our studies showthat the PrP promoter can also be used to create transgenic mice with Aβdeposits. Both the PDGF and PrP promoters drive βAPP expression chieflyin neurons of the cerebrum and cerebellum. Clearly, the βAPP variantwith K670N-M671L is effective, as is V717F, in promoting abundant plaquedeposition. The mice of Games and colleagues and Quon and colleagues(Quon, et al., Nature 352:239-41 (1991)) expressed largely orexclusively βAPP containing the KPI domain. We have demonstrated that aβAPP transgene lacking the KPI domain also is capable of engenderingamyloid plaques in mice.

These transgenic mice are unique in developing deficits in learning andmemory associated with elevated Aβ levels and the appearance of classicsenile plaques with dense amyloid cores. Whether the learning and memorydeficits in these mice are caused by or merely correlate with a rise inbrain Aβ levels and amyloid deposition remains unresolved. Furtherrefinements in temporal correlations between behavioral, biochemical,and histological changes in these transgenic mice may provide answers tothis fundamental question. The value of these mice resides in theircorrelative manifestation of learning and memory deficits, elevated Aβlevels, and amyloid plaques, providing new opportunities to study theelectrophysiology, pathophysiology, biochemistry, genetics, andneurobiology of AD.

Example 9 Testing for Drugs That Prevent Progressive Neurologic Disease

The animals of the invention are used to test materials for the abilityto confer protection against the development of progressive neurologicdisease. An animal exhibiting the progressive neurologic disease istreated with a test material in parallel with an untreated controltransgenic animal exhibiting the neurologic disease. A comparativelylower incidence of the progressive neurologic disease in the treatedanimal is detected as an indication of protection. Treated and untreatedanimals are analyzed for diminished exploratory/locomotor behavior (CItest; see Example 6), as well as diminished 2-deoxyglucoseuptake/utilization and hypertrophic gliosis in the cortico-limbicstructures of the brain. To determine if a treatment can prevent ordelay the onset of disease, half of the transgenic mice in a litter froma line of mice known to develop neurologic illness may be randomlyassigned to receive the treatment, and the other half to receive aplacebo, beginning at an age prior to the earliest known onset ofdisease for the given line of mice. The number of litters to be usedwill depend upon the magnitude of the differences observed betweentreated and untreated mice.

Mice are observed daily; their diagnosis is facilitated by the use ofthe CI test (see Example 6) which is administered three times per weekby individuals blinded to the experimental groups. Survival curves andmean ages of disease onset and death are calculated from the accumulatedclinical data.

Clinical results are corroborated by performing neuropathologic andglucose-uptake studies in samples in the experimental and controlgroups. Gliosis is evaluated in immunohistologic studies usingantibodies to glial fibrillary acidic protein. Glucose-uptake studiesare performed using a modification of the Sokoloff method described byChmielowska, et al., (1986) Exp. Brain Res. 63:607.

To determine if a treatment can ameliorate or cure disease, sicklittermates are randomly assigned to receive the treatment of interestor a saline placebo. Survival and clinical improvement on the CI testcoupled with neuropathologic and glucose-uptake studies are ascertained,as described above.

Example 10 Testing for Drugs That Cure Progressive Neurologic Disease

The animals of the invention are used to test materials for the abilityto improve or cure progressive neurologic disease. An animal exhibitingthe progressive neurologic disease is treated with a test material inparallel with an untreated control transgenic animal exhibiting theneurologic disease. A comparatively delayed death, or an improvement inthe neurobehavioral, pathologic, or functional indications of thedisease is detected as an indication of protection. Treated anduntreated animals are analyzed for diminished exploratory/locomotorbehavior, as well as diminished 2-deoxyglucose uptake/utilization andhypertrophis gliosis in the cortico-limbic structures of the brain.

As demonstrated by the above results, the clinical and pathologicfindings in non-human mammals with super endogenous levels of eithermutant or native amyloid precursor protein show an unexpected, butstriking parallel to these in humans with progressive neurologicdisorders such as Alzheimer's disease; the involved regions of theneocortex in affected transgenic mice and humans are similar. Inaddition, glucose uptake in the sensorimotor area of the cerebral cortexwas unaffected by the neurologic disease in transgenic mice. This wasthe only region of mouse neocortex sampled which represented mainlyprimary neocortex, rather than a mixture of primary and associationneocortex. It is a well-known observation that in brains of patientswith Alzheimer's disease, the primary neocortex is relatively free ofneuropathologic findings compared to the association cortex.

The CNS phenotype of the transgenic mice closely resembles the CNSphenotype of a subset of aged non-transgenic mice of the same FVBstrain. The gliosis in the hippocampus astrocytic gliosis that ischaracteristically found in the hippocampal formations of aged,memory-deficient rats (Landfield, et al. (1977) J. Gerontology 32:2-12)and aged, nude mice (Mandybur, et al., (1989) Acta Neuropathol (Berl.)77:507-513). The regional glucose hypometabolism in both the affectedtransgenic mice and the aged, impaired non-transgenic mice was markedlydiminished in the hippocampus, cerebral cortex, and amygdala, resemblingthe pattern of glucose hypometabolism occurring in humans with AD (deLeon, et al., (1983) Am. J. Neuroradiology 4:568-571), and in restrictedareas of the limbic system in aged, impaired Sprague-Dawley rats (Gage,et al., (1984) J. Neuroscience 11:2856-2865). The striking similaritiesin the neurologic disease exhibited by the transgenic animals and thenaturally occurring disorder in older mice of the same strain supportthe use of these transgenic mice as a model for progressive senescentdisorders of the brain, including Alzeheimer's disease.

Animals dying of neurologic disease exhibited hypertrophic gliosis inthe hippocampus, amygdala, and some areas of the cerebral cortex.Immunohistologic mapping of HuAPP in the transgenic mice indicatedwidespread expression throughout the brain. However, the behavioralabnormalities corresponded to the circumscribed regions of glioticpathology and glucose hypo-utilization found in select forebrainregions. The striking similarities in target cell specificities incortico-limbic areas of the brain (hippocampus, amygdala, and some areasof cerebral cortex) in these transgenic mice and Alzheimer's diseasesupport the use of these transgenic mice as a model for pprogressiveneurologic disorders such as Alzheimer's disease.

In summary, these transgenic mice express super-endogenous levels ofAPP. In the mouse lines which develop neurologic disease, APP transgeneproduct expression with at least 200% of endogenous levels has beenattained, or more than double that reported in any prior publications.More importantly, these mice have a definite, progressive neurologicdisorder. Even where APP expression has been achieved in othertransgenic mice, they have not developed a progressive disease affectingthe cortico-limbic areas of the brain. Transgenic mice (FVB/N)overexpressing wild-type and variant human or mouse βAPP695 develop acentral nervous system disorder involving cortico-limbic regions of thebrain sparing somatosensory-motor areas that resembles an acceleratednaturally occurring senescent disorder of FVB/N mice. Parameters thatinfluence the phenotype of transgenic mice expressing βAPP include hoststrain, βAPP primary structure, and levels of βAPP expression.Transgenic mice overexpressing the 695-amino acid isoform of humanK670N-M671L Alzheimer β-amyloid precursor protein (βAPP) have normallearning and memory in spatial reference and alternation tasks at threemonths of age but show impairment by nine to ten months of age. Afive-fold increase in Aβ1-40 and 14-fold increase in Aβ1-42(43)accompanied the appearance of these behavioral deficits. Numerouscongophilic Aβ plaques were present in cortical and limbic structures inmice with elevated Aβ levels. The correlative appearance of behavioral,biochemical and pathological abnormalities reminiscent of Alzheimer'sdisease (AD) affords new opportunities for exploring the pathophysiologyand neurobiology of AD in mice.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the are that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

17 9 base pairs nucleic acid single linear DNA (genomic) 1 GCGATGCTG 9 9base pairs nucleic acid single linear DNA (genomic) 2 ACCATGCTG 9 9 basepairs nucleic acid single linear DNA (genomic) 3 ACCATGGTG 9 9 basepairs nucleic acid single linear DNA (genomic) 4 ACGATGCTG 9 9 basepairs nucleic acid single linear DNA (genomic) 5 ATCATGGCG 9 31 basepairs nucleic acid single linear DNA (genomic) 6 AAGTCGACAC CATGCTGCCCGGTTTGGCAC T 31 24 base pairs nucleic acid single linear DNA (genomic) 7AAGGTACCTC CCAGCGCCCG AGCC 24 35 base pairs nucleic acid single linearDNA (genomic) 8 AAAAAAGTCG ACACCATGGT GCCCGGTTTG GCACT 35 23 base pairsnucleic acid single linear DNA (genomic) 9 AAGGTACCTC CAGCGCCCGA GCC 2327 base pairs nucleic acid single linear DNA (genomic) 10 CCGAGATCTCTGAAGTGAAG ATGGATG 27 27 base pairs nucleic acid single linear DNA(genomic) 11 AAGCTTGGCG CCTTTGTTTG AACCCAC 27 28 base pairs nucleic acidsingle linear DNA (genomic) 12 CCGAGATCTC TGAAGTGAAT CTGGATGC 28 25 basepairs nucleic acid single linear DNA (genomic) 13 CTGACCACTC GACCAGGTTCTGGGT 25 29 base pairs nucleic acid single linear DNA (genomic) 14GTGGATAACC CCTCCCCCAG CCTAGACCA 29 28 base pairs nucleic acid singlelinear DNA (genomic) 15 AAGCGGCCAA AGCCTGGAGG GTGGAACA 28 18 base pairsnucleic acid single linear DNA (genomic) 16 GTCGACACCA TGCTGCCC 18 12base pairs nucleic acid single linear DNA (genomic) 17 AACTAGCAGC TG 12

What is claimed is:
 1. A transgenic mouse whose genome comprises atransgene, said transgene having a prion gene promoter operably linkedto a sequence encoding an amyloid precursor protein (APP), said APPhaving at least one mutation associated with Alzheimer's disease,wherein said transgenic mouse produces amyloid plaques that aredetectable by Congo red staining in the brain of said transgenic mouse.2. The transgenic mouse of claim 1, wherein said at least one mutationcomprises the Swedish mutation.
 3. The transgenic mouse of claim 1,wherein said at least one mutation comprises a mutation at amino acid717.
 4. The transgenic mouse of claim 3, wherein a phenylalanine or aglycine residue is substituted for a valine at amino acid
 717. 5. Thetransgenic mouse of claim 1, wherein a non-transgenic ancestor of saidmouse is from a strain selected from the group consisting of SwissWebster and C57B6.
 6. Progeny of said transgenic mouse according toclaim 1, wherein the genomes of said progeny comprise said transgene,wherein said progeny exhibit amyloid plaques that are detectable byCongo red staining in the brain of said progeny.